Epoxyeicosatrienoic acid analogs and methods of making and using the same

ABSTRACT

Compounds and compositions comprising epoxyeicosatrienoic acid (EET) analogs that act as EET agonists and are useful as medications in the treatment of drug-induced nephrotoxicity, hypertension and other related conditions. Methods of making and using the compounds and compositions are further described.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DK38226, HL59699,GM31278, and HL51055 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application represents the national stage entry of PCTInternational Application No. PCT/US2012/032090 filed on Apr. 4, 2012and claims the benefit of U.S. Provisional Appl. No. 61/472,410 filedApr. 6, 2011, and U.S. Provisional Appl. No. 61/608,361 filed Mar. 8,2012, all of which are hereby incorporated by reference herein in theirentirety and for all purposes.

FIELD OF THE INVENTION

This invention relates generally to analogs of epoxyeicososatrienoicacid (EET). More particularly, the present invention is directed to EETanalogs that act as EET agonists and are useful as medications in thetreatment of drug-induced nephrotoxicity, hypertension and other relatedconditions.

BACKGROUND OF THE INVENTION

Epoxyeicosatrienoic acids (EETs) are signaling molecules that can act asshort-range hormones, (i.e. they are autocrine and paracrine mediators)of the cardiovascular system and kidney. They produce vasorelaxation aswell as anti-inflammatory and pro-fibrinolytic effects.

Hypertension and Related Conditions.

Cardiovascular disease afflicts 81 million of the 300 million people inthe United States, and 75 million of these people have hypertension. CYPepoxygenase metabolites have biological actions that implicate them asimportant contributors to cardiovascular function and blood pressurecontrol.

One of the first biological activities described for epoxyeicosatrienoicacids (EETs) was inhibition of renal tubular sodium reabsorption.Subsequently, EETs were determined to dilate blood vessels and wereidentified as endothelium-derived hyperpolarizing factors (EDHF). Thesebiological actions are consistent with the idea that EETs would beeicosanoids that contribute to lowering of blood pressure and preventsalt-sensitive hypertension.

Altered levels of EETs may contribute to hypertension in humans. Asingle nucleotide polymorphism in a CYP epoxygenase gene is associatedwith hypertension. Experimental studies in rodents have alsodemonstrated hypertension in conditions where kidney CYP epoxygenaseenzyme and/or EET levels were decreased. Increasing EET levels with11,12-EET-SI, a 11,12-EET analog, improved renal afferent arteriolarfunction in vitro.

Currently, soluble epoxide hydrolase inhibitors (sEHI) are used in vivoto increase EET levels and this results in a generalized increase in11,12-EET and 14,15-EET and to a lesser extent 8,9-EET. Recent in vivostudies have demonstrated that EET analogs lower blood pressure inhypertensive rats, and also ameliorate the metabolic syndrome phenotypein hemeoxygenase 2 deficient mice and prevent the adiposity-relatedvascular and renal damage. It does appear as if some of the EET agonistslike NUDSA may also inhibit sEH and increase CYP2C epoxygenaseexpression. This type of combinational activity described for NUDSAcould provide added beneficial effects. As a whole, these findings havegenerated interest in targeting the CYP epoxygenase pathway and EETs forthe treatment of hypertension.

Even though EETs have actions on renal tubular transport and vascularfunction that are essential for blood pressure regulation it has becomeapparent that additional biological actions ascribed to EETs made theman excellent therapeutic target for other cardiovascular diseases. Theseadditional activities demonstrated for EETs include inhibition ofplatelet aggregation and anti-inflammation. EETs also have been found tohave effects on vascular migration and proliferation, includingpromoting angiogenesis. Thus, EETs have become a therapeutic target forend organ damage associated with cardiovascular diseases, cardiacischemic injury, atherosclerosis, and stroke.

The therapeutic potential for EET agonists and sEHIs could extend beyondhypertension and cardiovascular diseases. Neural protection fromischemic injury has been attributed to sEHI actions on blood vessels andneurons. There is growing evidence that sEHIs provide protection fromischemic damage in the brain and heart through effects on apoptoticsignaling cascades. EET agonists and sEHIs have also been demonstratedto modulate pain in various experimental animal models. Other possibletherapeutic applications for EET agonists are sure to be discovered whenthese agents are tested in other disease models.

Accordingly, there is a need in the art for novel EET agonists that areactive as therapeutic agents against hypertension and relatedcardiovascular and neural disease.

Drug-Induced Nephrotoxicity.

A common side-effect of many drugs used in the treatment of variousconditions is nephrotoxicity. For instance, cisplatin, a platinum-basedinorganic compound, is one of the most potent and widely usedchemotherapy agents available to treat a variety of malignancies,including ovarian, lung, testicular and bladder cancers. Although,cisplatin is used as an important chemotherapy drug in the clinic, ithas potentially lethal adverse effects. The most common of this adverseeffect is nephrotoxicity (25-40% of cisplatin treated patients developacute renal failure), which limits the safe and effective use of thiswidely used chemotherapeutic agent. The pathophysiology ofcisplatin-induced nephrotoxicity involves enhanced oxidative stress,inflammation, increased endoplasmic reticulum (ER) stress and renal cellapoptosis.

EET is an important lipid mediator that exerts a number of biologicalactions including anti-inflammatory, anti-oxidative and anti-apoptoticactivities. A numbers of studies demonstrated that withanti-inflammatory, anti-apoptotic and anti-oxidative activities, EETpossess strong organ protective potential. For instance, increased EETbioavailability resulted from reduced conversion of EET to its lessactive form by soluble epoxide hydrolase (sEH) inhibitor provides kidneyprotection in a number of preclinical models of human diseases. Thesestudies demonstrated that the kidney protective effect of EET wasrelated to anti-inflammatory and anti-oxidative effects of EET. Indeed,there is strong evidence that EET have anti-inflammatory effects againstacute and chronic inflammation. Apart from inflammation, EET alsoprotect cells from apoptosis. Thus, there are strong evidences of EET'sability to protect organ by mechanisms involve its anti-inflammatory,anti-apoptotic and anti-oxidative activities.

However, it is known that endogenously produced EETs are chemically andmetabolically labile. Also, rapid metabolism, low solubility and storageissue limit the therapeutic prospect of EET. As such considerableinterest has arisen in developing strategies to enhance thebioavailability of EET. In this effort, attempts have been made todevelop EET analogs that possess EET-mimetic activity along with severalkey features important for stability and bio-availability. Several ofsuch EET analogs have demonstrated a number of biological activitiesincluding organ protection.

In the present study we have investigated the kidney protective effectof two newly developed orally active EET analogs in cisplatin-inducednephrotoxicity. We have demonstrated that EET analogs offered markedreno-protection during cisplatin administration and this effect wasrelated to their anti-oxidative, anti-inflammatory, anti-ER stress andanti-apoptotic activities. We have further demonstrated that whileprotecting the kidney from the deleterious nephrotoxic effects ofcisplatin, these EET analogs did not compromise cisplatin'schemotherapeutic effect.

Accordingly, there is a need in the art for novel EET analogs that areactive as therapeutic agents against the deleterious nephrotoxic effectsof cisplatin.

SUMMARY OF THE INVENTION

Here, the inventors demonstrate novel compositions ofepoxyeicosatrienoic acids (EET) analogs and methods of use thereof forthe treatment of cardiovascular disease, particularly the use of suchcompositions as anti-hypertensive agents.

Accordingly, the invention encompasses in a first aspect certaincompounds that are 14,15-EET analogs. In certain embodiments, thecompound has the structure of any one of the following compounds.

In one embodiment, the invention comprises compounds 7 and 30.

In another aspect, the invention provides a method of making any ofcompounds 1-33 as described and claimed herein.

Compounds according to the invention are, in certain embodiments,provided in the form of a composition comprising a compound as describedand claimed herein in combination with a pharmaceutically acceptablecarrier.

The present invention further encompasses methods of providing treatmentof hypertension in a subject, resulting in a reduction of blood pressurein a subject. Such methods include steps of administering to a subject atherapeutically effective amount of any of compounds 1-33, alone or incombination, as described and claimed herein, whereby blood pressure inthe subject is reduced. In one embodiment, the method comprisesadministering compound 7. In an alternate embodiment the methodcomprises administering compound 30.

In another embodiment, the present invention provides EET analogs havingthe structures selected from the group consisting of

In yet another embodiment, the invention encompasses the use of any ofthe 14,15-EET analogs described above for the manufacture of amedicament for treating hypertension in a subject. As well, the presentinvention further contemplates compounds according to the invention foruse in treating hypertension in a subject.

The present invention further encompasses methods of treatingdrug-induced nephrotoxicity in a subject. In one embodiment, theinvention comprises providing treatment for the deleterious nephrotoxiceffects of cisplatin in a subject. Such methods include steps ofadministering to a subject a therapeutically effective amount of acompound as described and claimed herein, whereby the deleteriousnephrotoxic effects of the drug in the subject are reduced.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the chemical structure SRD-I-71-9, which corresponds toEET analog compound 26 of Table 1. FIG. 1B shows mean measured bloodpressure as a function of days of treatment in spontaneouslyhypertensive rats administered a composition containing vehicle orcompound 26. FIG. 1C shows mean measured blood pressure at day 0, day 7,and day 14 in spontaneously hypertensive rats administered a compositioncontaining vehicle or compound 26.

FIG. 2A depicts the chemical structure LGK-I-119-15, which correspondsto EET analog compound 20 of Table 1. FIG. 2B shows mean measured bloodpressure as a function of days of treatment in spontaneouslyhypertensive rats administered a composition containing vehicle orcompound 20. FIG. 2C shows mean measured blood pressure at day 0, day 7,and day 14 in spontaneously hypertensive rats administered a compositioncontaining vehicle or compound 20.

FIG. 3A depicts the chemical structure JLJ-I-94-6, which corresponds toEET analog compound 7 of Table 1. FIG. 3B shows mean measured bloodpressure as a function of days of treatment in spontaneouslyhypertensive rats administered a composition containing vehicle orcompound 7. The data is graphed as 12 hour averages. Compounds weredelivered i.p. for 14 days. FIG. 3C shows mean measured blood pressureat day 0, day 7, and day 14 relative to initial treatment inspontaneously hypertensive rats administered a composition containingvehicle or compound 7.

FIG. 4A depicts the chemical structure MV-IV-110-20, which correspondsto EET analog compound 30 of Table 1. FIG. 4B shows mean measured bloodpressure as a function of days of treatment in spontaneouslyhypertensive rats administered a composition containing vehicle orcompound 30. The data is graphed as 12 hour averages. FIG. 3C shows meanmeasured blood pressure at day 0, day 7, and day 14 in spontaneouslyhypertensive rats administered a composition containing vehicle orcompound 30.

FIG. 5A shows mean measured blood pressure as a function of days oftreatment inangiotensin II induced hypertensive rats administered acomposition containing vehicle or compound 7. The data is graphed as 12hour averages. FIG. 5B shows mean measured blood pressure at day 0, day7, and day 14 in angiotensin II induced hypertensive rats administered acomposition containing vehicle or compound 7.

FIG. 6A shows mean measured blood pressure as a function of days oftreatment in angiotensin II induced hypertensive rats administered acomposition containing vehicle or compound 30. The data is graphed as 12hour averages. FIG. 6B shows mean measured blood pressure at day 0, day7, and day 14 in angiotensin II induced hypertensive rats administered acomposition containing vehicle or compound 30.

FIG. 7: (a) Plasma creatinine, (b) Blood urea nitrogen (BUN), (c) kidneyinjury molecule-1, and (d) urinary NA Gin cisplatin administered ratspretreated with either EET analogs, EET-A and EET-B or vehicle. *p<0.05vs. normal Wistar Kyotorat; #p<0.05 vs. vehicle treated rat administeredcisplatin. Data expressed as mean±SEM,n=5-7.

FIG. 8A: Representative photomicrographs of Periodicacid-Schiff (PAS)Staining (200×) depicting tubular cast formation along with thecalculated cast are a fraction (%) in the renal cortical sections ofdifferent experimental groups. *p<0.05 vs. normal Wistar Kyotorat;#p<0.05 vs. vehicle treated rat. Data expressed as mean±SEM, n=5-7.

FIG. 8B: Representative photomicrographs of Periodicacid-Schiff (PAS)Staining 200×) depicting tubular cast formation along with thecalculated cast area fraction (%) in the renal medullary sections ofdifferent experimental groups. *p<0.05 vs. normal Wistar Kyoto rat;#p<0.05 vs. vehicle treated rat. Data expressed as mean±SEM,n=5-7.

FIG. 9: RT-PCR analysis form RNA expressions of (a) NOX1, (b) gp91Phox,(d) SOD1, (e) SOD2, (f) SOD3 and (c) measurements of kidneythiobarbituric acid-reactive substances (TBARS) in cisplatinadministered rats treated with either EET analogs A, B or vehicle.*p<0.05 vs. normal Wistar Kyoto rat; #p<0.05 vs. vehicle pretreated ratadministered cisplatin. Data expressed as mean±SEM,n=5-7.

FIG. 10: Renal expression of inflammatory marker genes TNF-α (a), IL-6(b) and IL-1β (c) in cisplatinadministered rats pretreated with eitherEET analogs EET-A and EET-B or vehicle. *p<0.05 vs. normal Wistar Kyotorat; #p<0.05 vs. vehicle pretreated rat administered cisplatin. Dataexpressed as mean±SEM, n=5-7.

FIG. 11: Renal expression of endoplasmic reticulum stress marker genesGRP78/BiP(a) and caspase12 (b) in cisplatin administered rats treatedwith either EET analogs EET-A and EET-B or vehicle. *p<0.05 vs. normalWistarKyoto rat; #p<0.05 vs. vehicle treated rat administered. Dataexpressed as mean±SEM, n=5-7.

FIG. 12: Renal cortical caspase 3 activity (a) andrenal expression ofanti-apoptotic gene Bcl2 (b) in different experimental groups. Theratios between the renal expression of anti-apoptotic gene Bcl2 and theapoptotic genes Bak (c) and Bax (d) in different experimental groups.*p<0.05 vs. normal Wistar Kyoto rat; #p<0.05 vs. vehicle treated ratadministered cisplatin. Data expressed as mean±SEM, n=5-7.

FIG. 13A: Cytotoxic effect of cisplatin in normal kidney cell (HEK293)and cancer cells (Hela, U87, NCCIT).

FIG. 13B: Effect of EET analog EET-A on the cell growth of HEK293, Hela,U87, NCCIT. EET-A does not effect the chemotherapeutic effect ofcisplatin in NCCIT cancer cells. Data expressed as mean±SEM, n=5-7.

FIG. 14: Structure of EET analogs EET-A and EET-B.

FIG. 15: Synthesis of EET-B.

FIG. 16A: Synthesis of EET-A.

FIG. 16B: Alternate synthesis of EET-A.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby any later-filed nonprovisional applications.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

II. The Invention

The inventors here disclose novel EET analogs, EET agonists, and otherrelated lipid compounds, and compositions comprising such compounds, aswell as methods of synthesizing such compounds and the use of suchcompositions in treating hypertension and related conditions in treatingthe deleterious effects of cisplatin nephrotoxicity and relatedconditions. The inventors' have shown that several of the compoundsexhibit anti-hypertensive effects and are well-tolerated in relevant ratmodels. A number of different delivery options are possible, includingintraperitoneal injections, blood injections, or oral delivery.Liposomes, mycelles, and emulsifiers can be used in to make thesepreparations more soluble.

As used herein, “subject” means mammals and non-mammals. “Mammals” meansany member of the class Mammalia including, but not limited to, humans,non-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, horses, sheep, goats, and swine;domestic animals such as rabbits, dogs, and cats; laboratory animalsincluding rodents, such as rats, mice, and guinea pigs; and the like.Examples of non-mammals include, but are not limited to, birds, and thelike. The term “subject” does not denote a particular age or sex.

As used herein, “administering” or “administration” includes any meansfor introducing a compound of the present invention into the body,preferably into the systemic circulation. Examples include but are notlimited to oral, buccal, sublingual, pulmonary, transdermal,transmucosal, as well as subcutaneous, intraperitoneal, intravenous, andintramuscular injection.

A “therapeutically effective amount” means an amount of a compound that,when administered to a subject for treating a disease or condition, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease state being treated, the severity or the disease treated,the age and relative health of the subject, the route and form ofadministration, the judgment of the attending medical or veterinarypractitioner, and other factors.

For purposes of the present invention, “treating” or “treatment”describes the management and care of a patient for the purpose ofcombating the disease, condition, or disorder. The terms embrace bothpreventative, i.e., prophylactic, and palliative treatments. Treatingincludes the administration of a compound of present invention toprevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder.

A compound is administered to a patient in a therapeutically effectiveamount. A compound can be administered alone or as part of apharmaceutically acceptable composition. In addition, a compound orcomposition can be administered all at once, as for example, by a bolusinjection, multiple times, such as by a series of tablets, or deliveredsubstantially uniformly over a period of time, as for example, usingtransdermal delivery. Further, the dose of the compound can be variedover time. A compound can be administered using an immediate releaseformulation, a controlled release formulation, or combinations thereof.The term “controlled release” includes sustained release, delayedrelease, and combinations thereof.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a patient or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the human treated and further depending upon theroute by which the composition is to be administered. By way of example,the composition can comprise between 0.1% and 100% (w/w) activeingredient. A unit dose of a pharmaceutical composition of the inventionwill generally comprise from about 100 milligrams to about two grams ofthe active ingredient, and preferably comprises from about 200milligrams to about 1.0 gram of the active ingredient.

A preferred dosage for humans would be in the low mg/kg rangeadministered orally once daily. Twice daily would also be acceptable.

To improve water solubility, the preferred compounds can be formulatedwith cyclodextrins or cyclodextrin-derived products, derivatized withsubstituents such as polyethylene glycols or other polar functionality,or included in liposomes. For oral delivery, the compounds may bemodified with lipophilic functionality or conjugated to activelyabsorbed molecules. Other approaches are discussed in “Strategies toimprove oral drug bioavailability”, Isabel Gomez-Orellana, ExpertOpinion on Drug Delivery, May 2005, Vol. 2, No. 3: Pages 419-433, whichis incorporated by reference herein.

Another aspect of the invention relates to a kit comprising apharmaceutical composition of the invention and instructional material.Instructional material includes a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the pharmaceutical composition of the invention for one ofthe purposes set forth herein in a human. The instructional material canalso, for example, describe an appropriate dose of the pharmaceuticalcomposition of the invention. The instructional material of the kit ofthe invention can, for example, be affixed to a container which containsa pharmaceutical composition of the invention or be shipped togetherwith a container which contains the pharmaceutical composition.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thepharmaceutical composition be used cooperatively by the recipient.

The invention also includes a kit comprising a pharmaceuticalcomposition of the invention and a delivery device for delivering thecomposition to a human. By way of example, the delivery device can be asqueezable spray bottle, a metered-dose spray bottle, an aerosol spraydevice, an atomizer, a dry powder delivery device, a self-propellingsolvent/powder-dispensing device, a syringe, a needle, a tampon, or adosage-measuring container. The kit can further comprise aninstructional material as described herein. The kit also comprises acontainer for the separate compositions, such as a divided bottle or adivided foil packet. Additional examples of containers include syringes,boxes, bags, and the like. Typically, a kit comprises directions for theadministration of the separate components. The kit form is particularlyadvantageous when the separate components are preferably administered indifferent dosage forms (e.g., oral and parenteral), are administered atdifferent dosage intervals, or when titration of the individualcomponents of the combination is desired by the prescribing physician.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen that the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday,” etc. Othervariations of memory aids will be readily apparent. A “daily dose” canbe a single tablet or capsule or several pills or capsules to be takenon a given day.

In another embodiment of the present invention, a dispenser designed todispense the daily doses one at a time in the order of their intendeduse is provided. Preferably, the dispenser is equipped with a memoryaid, so as to further facilitate compliance with the dosage regimen. Anexample of such a memory aid is a mechanical counter, which indicatesthe number of daily doses that have been dispensed. Another example ofsuch a memory aid is a battery-powered micro-chip memory coupled with aliquid crystal readout, or audible reminder signal which, for example,reads out the date that the last daily dose has been taken and/orreminds one when the next dose is to be taken.

The compounds of the present invention, optionally comprising otherpharmaceutically active compounds, can be administered to a patienteither orally, rectally, parenterally, (for example, intravenously,intramuscularly, or subcutaneously) intracisternally, intravaginally,intraperitoneally, intravesically, locally (for example, powders,ointments or drops), or as a buccal or nasal spray. Other contemplatedformulations include projected nanoparticles, liposomal preparations,resealed erythrocytes containing the active ingredient, andimmunologically-based formulations.

Parenteral administration of a pharmaceutical composition includes anyroute of administration characterized by physical breaching of a tissueof a human and administration of the pharmaceutical composition throughthe breach in the tissue. Parenteral administration thus includesadministration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection and intravenous,intraarterial, or kidney dialytic infusion techniques. For example, thecompositions of the present invention can be administered to a subjectby brain (via vPAG) injections, intrathecal injections, intraperitonealinjections, or blood injections.

Compositions suitable for parenteral injection comprise the activeingredient combined with a pharmaceutically acceptable carrier such asphysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, or may comprise sterile powdersfor reconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, isotonic saline, ethanol, polyols(propylene glycol, polyethylene glycol, glycerol, and the like),suitable mixtures thereof, triglycerides, including vegetable oils suchas olive oil, or injectable organic esters such as ethyl oleate. Properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and/or by the use of surfactants. Such formulations canbe prepared, packaged, or sold in a form suitable for bolusadministration or for continuous administration. Injectable formulationscan be prepared, packaged, or sold in unit dosage form, such as inampules, in multi-dose containers containing a preservative, or insingle-use devices for auto-injection or injection by a medicalpractitioner.

Formulations for parenteral administration include suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchformulations can further comprise one or more additional ingredientsincluding suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the activeingredient is provided in dry (i.e. powder or granular) form forreconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and can comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides. Otherparentally-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystems. Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

The compounds according to the present invention may also containadjuvants such as preserving, wetting, emulsifying, and/or dispersingagents, including, for example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example, sugars, sodium chloride, and the like. Prolonged absorptionof injectable pharmaceutical compositions can be brought about by theuse of agents capable of delaying absorption, for example, aluminummonostearate and/or gelatin. In particular, liposomes, mysomes andemulsifiers can be used in to make the present compounds more solublefor delivery.

Dosage forms can include solid or injectable implants or depots. Inpreferred embodiments, the implant comprises an effective amount of anactive agent and a biodegradable polymer. In preferred embodiments, asuitable biodegradable polymer can be selected from the group consistingof a polyaspartate, polyglutamate, poly(L-lactide), a poly(D,L-lactide),a poly(lactide-co-glycolide), a poly(s-caprolactone), a polyanhydride, apoly(beta-hydroxy butyrate), a poly(ortho ester) and a polyphosphazene.In other embodiments, the implant comprises an effective amount ofactive agent and a silastic polymer. The implant provides the release ofan effective amount of active agent for an extended period of about oneweek to several years.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage form, the active compound isadmixed with at least one inert customary excipient (or carrier) such assodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, or silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, or sodium carbonate; (e) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol or glycerol monostearate; (h) adsorbents, as for example, kaolinor bentonite; and/or (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules and tablets, thedosage forms may also comprise buffering agents.

A tablet comprising the active ingredient can, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture.

Pharmaceutically acceptable excipients used in the manufacture oftablets include inert diluents, granulating and disintegrating agents,binding agents, and lubricating agents. Known dispersing agents includepotato starch and sodium starch glycolate. Known surface active agentsinclude sodium lauryl sulfate. Known diluents include calcium carbonate,sodium carbonate, lactose, microcrystalline cellulose, calciumphosphate, calcium hydrogen phosphate, and sodium phosphate. Knowngranulating and disintegrating agents include corn starch and alginicacid. Known binding agents include gelatin, acacia, pre-gelatinizedmaize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.Known lubricating agents include magnesium stearate, stearic acid,silica, and talc.

Tablets can be non-coated or they can be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of a human,thereby providing sustained release and absorption of the activeingredient. By way of example, a material such as glyceryl monostearateor glyceryl distearate can be used to coat tablets. Further by way ofexample, tablets can be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets. Tablets can further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Solid dosage forms such as tablets, dragees, capsules, and granules canbe prepared with coatings or shells, such as enteric coatings and otherswell known in the art. They may also contain opacifying agents, and canalso be of such composition that they release the active compound orcompounds in a delayed manner. Examples of embedding compositions thatcan be used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike. Hard capsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and can further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin. Soft gelatincapsules comprising the active ingredient can be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which can be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Oral compositions can be made, using known technology, whichspecifically release orally-administered agents in the small or largeintestines of a human patient. For example, formulations for delivery tothe gastrointestinal system, including the colon, include enteric coatedsystems, based, e.g., on methacrylate copolymers such aspoly(methacrylic acid, methyl methacrylate), which are only soluble atpH 6 and above, so that the polymer only begins to dissolve on entryinto the small intestine. The site where such polymer formulationsdisintegrate is dependent on the rate of intestinal transit and theamount of polymer present. For example, a relatively thick polymercoating is used for delivery to the proximal colon (Hardy et al.,Aliment. Pharmacol. Therap. (1987) 1:273-280). Polymers capable ofproviding site-specific colonic delivery can also be used, wherein thepolymer relies on the bacterial flora of the large bowel to provideenzymatic degradation of the polymer coat and hence release of the drug.For example, azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friendet al., J. Med. Chem. (1984) 27:261-268) and a variety of naturallyavailable and modified polysaccharides (see PCT applicationPCT/GB89/00581) can be used in such formulations.

Pulsed release technology such as that described in U.S. Pat. No.4,777,049 can also be used to administer the active agent to a specificlocation within the gastrointestinal tract. Such systems permit drugdelivery at a predetermined time and can be used to deliver the activeagent, optionally together with other additives that my alter the localmicroenvironment to promote agent stability and uptake, directly to thecolon, without relying on external conditions other than the presence ofwater to provide in vivo release.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage form may containinert diluents commonly used in the art, such as water or othersolvents, isotonic saline, solubilizing agents and emulsifiers, as forexample, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, almond oil, arachis oil,coconut oil, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame seed oil, MIGLYOL™, glycerol, fractionated vegetableoils, mineral oils such as liquid paraffin, tetrahydrofurfuryl alcohol,polyethylene glycols, fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the compounds of the present invention canalso include adjuvants, such as wetting agents, emulsifying andsuspending agents, demulcents, preservatives, buffers, salts,sweetening, flavoring, coloring and perfuming agents. Suspensions, inaddition to the active compound, may contain suspending agents, as forexample, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol orsorbitan esters, microcrystalline cellulose, hydrogenated edible fats,sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia,agar-agar, and cellulose derivatives such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,aluminum metahydroxide, bentonite, or mixtures of these substances, andthe like. Liquid formulations of a pharmaceutical composition of theinvention that are suitable for oral administration can be prepared,packaged, and sold either in liquid form or in the form of a dry productintended for reconstitution with water or another suitable vehicle priorto use.

Known dispersing or wetting agents include naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include lecithin and acacia.Known preservatives include methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

Liquid solutions of the active ingredient in aqueous or oily solventscan be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention can comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Compositions for rectal or vaginal administration can be prepared bymixing a compound of the present invention and any additional compoundswith suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax, which are solid atordinary room temperature, but liquid at body temperature, andtherefore, melt in the rectum or vaginal cavity and release the activeingredient. Such a composition can be in the form of, for example, asuppository, a retention enema preparation, and a solution for rectal orcolonic irrigation. Suppository formulations can further comprisevarious additional ingredients including antioxidants and preservatives.Retention enema preparations or solutions for rectal or colonicirrigation can be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is known in the art,enema preparations can be administered using, and can be packagedwithin, a delivery device adapted to the rectal anatomy of a human.Enema preparations can further comprise various additional ingredientsincluding antioxidants and preservatives.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition can be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Dosage forms for topical administration of a compound according to thepresent invention include ointments, powders, sprays and inhalants. Thecompounds are admixed under sterile conditions with a physiologicallyacceptable carrier, and any preservatives, buffers, and/or propellantsthat may be required. Formulations suitable for topical administrationinclude liquid or semi-liquid preparations such as liniments, lotions,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes, and solutions or suspensions. Topically-administrableformulations can, for example, comprise from about 0.1% to about 10%(w/w) active ingredient, although the concentration of the activeingredient can be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration canfurther comprise one or more of the additional ingredients describedherein.

Ophthalmic formulations, eye ointments, powders, and solutions are alsocontemplated as being within the scope of this invention. Suchformulations can, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops can furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. In other embodiments, ophthalmalmicallyadministrable formulations comprise the active ingredient inmicrocrystalline form or in a liposomal preparation.

Pharmaceutical compositions of the invention formulated for pulmonarydelivery can provide the active ingredient in the form of droplets of asolution or suspension. Such formulations can be prepared, packaged, orsold as aqueous or dilute alcoholic solutions or suspensions, optionallysterile, comprising the active ingredient, and can conveniently beadministered using any nebulization or atomization device. Suchformulations can further comprise one or more additional ingredientsincluding a flavoring agent such as saccharin sodium, a volatile oil, abuffering agent, a surface active agent, or a preservative such asmethylhydroxybenzoate. The droplets provided by this route ofadministration preferably have an average diameter in the range fromabout 0.1 to about 200 nanometers.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations can, for example, be in the form of tablets or lozengesmade using conventional methods, and can, for example, comprise 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration can comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or atomized formulations, whendispersed, preferably have an average particle or droplet size in therange from about 0.1 to about 200 nanometers, and can further compriseone or more of the additional ingredients described herein.

For parenteral administration in non-human animals, the compounds of thepresent invention may be prepared in the form of a paste or a pellet andadministered as an implant, usually under the skin of the head or ear ofthe animal. Paste formulations can be prepared by dispersing a compoundor compounds in pharmaceutically acceptable oil such as peanut oil,sesame oil, corn oil or the like. Pellets containing a therapeuticallyeffective amount of a compound or compounds can be prepared by admixingthe compound with a diluent such as a carbowax, carnauba wax, and thelike, and a lubricant, such as magnesium or calcium stearate, can beadded to improve the pelleting process. It is, of course, recognizedthat more than one pellet may be administered to an animal to achievethe desired dose level. Moreover, it has been found that such implantsmay also be administered periodically during the animal treatment periodin order to maintain the proper active agent level in the animal's body.

The compounds of the present invention and the pharmaceuticallyacceptable salts of the same, can be administered to a patient at dosagelevels in the range of from about 0.01 to about 1,000 mg per day. For anormal adult human having a body weight of about 70 kg, a dosage in therange of from about 0.01 to about 300 mg is typically sufficient, with1-10 mg/kg a preferred dosage. However, some variability in the generaldosage range may be required depending upon the age and weight of thesubject being treated, the intended route of administration, theparticular compound being administered and the like. The determinationof dosage ranges and optimal dosages for a particular patient is wellwithin the ability of one of ordinary skill in the art having thebenefit of the instant disclosure. It is also noted that the compoundsof the present invention can be used in sustained release, controlledrelease, and delayed release formulations, which forms are also wellknown to one of ordinary skill in the art.

It is not critical whether the compounds of the present invention areadministered directly to the cell, to a tissue comprising the cell, abody fluid that contacts the cell, or a body location from which thecompound can diffuse or be transported to the cell. It is sufficientthat the compound is administered to the patient in an amount and by aroute whereby an amount of the compound sufficient to mobilize lipids inthe cell arrives, directly or indirectly at the cell. The minimum amountvaries with the identity of the compounds.

The specific dosage and dosage range that can be used depends on anumber of factors, including the requirements of the patient, theseverity of the condition being treated, and the pharmacologicalactivity of the compound being administered. The determination of dosageranges and optimal dosages for a particular patient is well within theordinary skill of one in the art in view of this disclosure. It isunderstood that the ordinarily skilled physician or veterinarian willreadily determine and prescribe an effective amount of the compound tomobilize lipid stores, induce weight loss, or inhibit appetite in thepatient. In so proceeding, the physician or veterinarian can, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. It isfurther understood, however, that the specific dose level for anyparticular human will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the human, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the severity of any disorder being treated.

Various exemplary embodiments of compositions and methods according tothis invention are now described in the following examples. In theseembodiments, specific products identified by Arabic numerals (e.g., 1,2, 3, etc.) refer to the specific structures so identified in thefollowing description, particularly in Table 1 below and the appendedclaims

III. EXAMPLES

The following examples are offered for illustrative purposes only andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

Example 1 Synthesis of 33 EET Analogs

In this Example, the inventors report the synthesis of a library of EETanalogs. The chemical structures of these compounds, designated ascompounds I-33, are shown in Table 1 below.

General Procedures.

Unless stated otherwise, yields refer to purified products and are notoptimized. Final compounds were judged ≧95% pure by HPLC using a ZorbaxEclipse C18 (250×4.6 mm; Agilent) connected to an Agilent 1200 API/LC-MSwith acetonitrile/water combinations as solvent. All oxygen and/ormoisture sensitive reactions were performed under an argon atmosphereusing oven-dried glassware and anhydrous solvents. Anhydrous solventswere freshly distilled from sodium benzophenone ketyl, except forCH₂Cl₂, which was distilled from Call₂. Extracts were dried overanhydrous Na₂SO₄ and filtered prior to removal of all volatiles underreduced pressure. Unless otherwise noted, commercially availablematerials were used without purification. Flash chromatography (FC) wasperformed using E Merck silica gel 60 (240-400 mesh). Thin layerchromatography (TLC) was performed using pre-coated plates purchasedfrom E. Merck (silica gel 60 PF254, 0.25 mm). Nuclear magnetic resonance(NMR) spectra were recorded on Varian 300, 400 or 500 spectrometers atoperating frequencies of 300/400/500 MHz (¹H) or 75/100/125 MHz (¹³C) inCDCl₃, unless otherwise stated. Nuclear magnetic resonance (NMR)splitting patterns are described as singlet (s), doublet (d), triplet(t), quartet (q), and broad (br); the values of chemical shifts (δ) aregiven in ppm relative to residual solvent (chloroform δ=7.27 for ¹H NMRor δ=77.23 for proton decoupled ¹³C NMR), and coupling constants (J) aregiven in Hertz (Hz). Melting points were determined using an OptiMelt(Stanford Research Systems) and are uncorrected. The Notre DameUniversity Mass Spectroscopy Facility or Prof. Kasem Nithipatikom(Medical College of Wisconsin) kindly provided high-resolution massspectral analyses.

TABLE 1 33 EET analogs and measured vascular relaxation and sEHinhibition activity. Vascular Relax. sEHi % EC₅₀ IC₅₀ Compd Analog (10μM) (μM) (μM) 1

86 2.9 >500 2

72 5.1 255 3

104 1.5 >500 4

49 0.63 237 5

95 1.9 >500 6

92 2.75 >500 7

91 1.6 392 8

73 6.0 41 9

61 6.7 71 10

71 >10 >500 11

63 7.6 57 12

75 3.4 32 13

48 >10 6 14

76 5.0 11 15

85 3.7 22 16

53 9.8 32 17

92 3.5 96 18

92 3.1 23 19

74 1.0 >500 20

119 0.18 11 21

96 1.7 >500 22

110 1.1 32 23

89 1.1 65 24

47 >10 57 25

109 0.34 10 26

109 0.32 >500 27

116 0.36 >500 28

93 3.3 31 29

54 2.4 231 30

96 1.3 >500 31

73 0.9 >500 32

96 2.4 >500 33

73 3.3 282

The synthesis of the EET-compounds of Table 1 are provided as follows:

Synthesis of Analog 25

tert-Butyldiphenyl-[12-(tetrahydro-2H-pyran-2-yloxy)dodec-7-ynyloxy)]silane

N-Butyllithium (12.0 mL of 2.5 M solution in hexanes, 30.0 mmol) wasadded dropwise with stirring to a −78° C. solution of2-(hex-5-ynyloxy)tetrahydro-2H-pyran (5.0 g, 27.43 mmol, G. F. SmithChem. Co.) in THF/HMPA (4:1, 150 mL) under an argon atmosphere. After 30min, the reaction mixture was warmed to 0° C. and maintained at thistemperature for 2 h. After re-cooling to −78° C., a solution of1-tert-butyldiphenylsilyloxy-6-bromohexane¹ (11.50 g, 27.43 mmol) in THF(55 mL) was added and the temperature was raised over 3 h to 23° C.After an additional 12 h, the reaction mixture was quenched withsaturated aq. NH₄Cl solution (25 mL). The mixture was extracted withEtOAc (2×100 mL) and the combined extracts were washed with water (2×150mL), brine (50 mL), dried, and concentrated under reduced pressure. Theresidue was purified by SiO₂ column chromatography to give the titlecompound (11.14 g, 78%), obtained as a colorless oil, whose spectraldata matched literature values. TLC: 15% EtOAc/hexanes, R_(f)˜0.60; ¹HNMR (400 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 4.57 (t, J=4.3 Hz,1H), 3.78-3.86 (m, 2H), 3.65 (t, J=6.3 Hz, 2H), 3.32-3.54 (m, 2H),2.10-2.22 (m, 4H), 1.24-1.84 (m, 18H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ130.61, 129.17, 124.54, 122.62, 93.82, 75.48, 74.89, 72.41, 72.10,71.78, 62.11, 58.93, 57.32, 27.51, 25.79, 24.18, 23.99, 23.68, 21.96,21.87, 21.0, 20.55, 20.40, 14.26, 13.77, 13.67.

12-(tert-Butyldiphenylsilyloxy)dodec-5-yn-1-ol

A mixture oftert-butyldiphenyl-[12-(tetrahydro-2H-pyran-2-yloxy)dodec-7-ynyloxy)]silane(11.0 g, 21.14 mmol) and p-toluenesulfonic acid (165 mg) in MeOH (110mL) was stirred at room temperature for 10 h. The reaction mixture wasquenched with sat. aq. NaHCO₃ solution (10 mL). The methanol wasevaporated, then more water (50 mL), and the mixture extracted withEtOAc (3×75 mL). The combined organic extracts were washed with water(2×50 mL), brine (40 mL), dried and concentrated under reduced pressure.The residue was purified by SiO₂ chromatography to give the titlecompound (7.93 g, 86%), obtained as a colorless oil, whose spectral datamatched literature values.¹ TLC: EtOAc/hexanes (3:7), R_(f)˜0.44; ¹H NMR(300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.62 (t, J=6.3 Hz,4H), 2.06-2.22 (m, 4H), 1.50-1.64 (m, 12H), 1.04 (s, 9H); ¹³C NMR (100MHz) δ 135.81, 134.36, 129.74, 127.82, 80.89, 80.01, 64.14, 62.71,32.71, 32.10, 29.34, 28.86, 27.11, 25.59, 25.57, 19.46, 18.93, 18.77.

12-(tert-Butyldiphenylsilyloxy)dodec-5(Z)-en-1-ol

NaBH₄ (82 mg, 2.28 mmol) was added in portions with vigorously stirringto a room temperature solution of Ni(OAc)₂.4H₂O (567 mg, 2.28 mmol) inabsolute ethanol (20 mL) under a hydrogen atmosphere (1 atm). After 15min, freshly distilled ethylenediamine (0.30 mL, 4.56 mmol) was added tothe black suspension, followed after a further 15 min by a solution of12-(tert-butyldiphenylsilyloxy)dodec-5-yn-1-ol (4.0 g, 9.16 mmol) inabsolute EtOH (10 mL). After 1 h, the reaction mixture was quenched withEt₂O (20 mL) and passed through a small bed of silica gel. The bed wasrinsed with another portion of Et₂O (5 mL). The combined etherealfiltrates were concentrated under reduced pressure to afford the titlecompound (3.85 g, 96%) as a colorless oil sufficiently pure to be useddirectly in the next step. TLC: EtOAc/hexanes (3:7), R_(f)˜0.46. ¹H NMR(300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 5.42-5.28 (m, 2H),3.65-3.60 (t, J=6.4 Hz, 4H), 2.08-1.96 (m, 4H), 1.50-1.60 (m, 4H),1.40-1.24 (m, 10H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ 135.81, 134.40,130.61, 129.71, 129.60, 127.80, 64.21, 63.14, 32.78, 32.60, 29.98,29.27, 27.42, 27.14, 27.10, 26.08, 25.92, 19.48. HRMS calcd forC₂₈H₄₃O₂Si [M+1]⁺ 439.3032. found 439.3027.

1-tert-Butyldiphenylsilyloxy-12-azidododec-7(Z)-ene

Diisopropyl azodicarboxylate (DIAD; 1.46 mL, 7.35 mmol) was addeddropwise to a −20° C. solution of PPh₃ (2.10 g, 8.0 mmol) in dry THF (45mL) under an argon atmosphere. After 10 min, a solution of12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-en-1-ol (3.20 g, 7.35 mmol)from above in dry THF (10 mL) was added dropwise. After 30 min, themixture was warmed to 0° C. and diphenylphosphoryl azide (1.58 mL, 7.35mmol) was added dropwise. After stirring 4 h at rt, the reaction mixturewas quenched with water (150 mL) and extracted with EtOAc (2×100 mL).The combined organic extracts were washed with brine (100 mL), dried(Na₂SO₄), and concentrated under reduced pressure. The residue waspurified by SiO₂ column chromatography eluting with 4% EtOAc/hexane toafford the title compound (2.45 g, 72%). TLC: EtOAc/hexanes (1:9),R_(f)˜0.55; ¹H NMR (400 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H),5.28-5.42 (m, 2H), 3.70 (t, J=5.8 Hz, 2H), 3.27 (t, J=6.3 Hz, 2H),1.96-2.10 (m, 4H), 1.24-1.64 (m, 12H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ135.84, 134.41, 130.93, 129.75, 129.12, 127.83, 64.22, 51.62, 32.81,29.93, 29.30, 28.68, 27.46, 27.14, 27.02, 26.90, 25.96, 19.49; IR (neat)2930, 2783, 2331, 2097, 1106 cm¹. HRMS calcd for C₂₈H₄₂N₃OSi [M+1]⁺464.3097. found 464.3099.

1-tert-Butyldiphenylsilyloxy-12-aminododec-7(Z)-ene

Triphenylphosphine (1.18 g, 4.50 mmol) was added to a stirring solutionof azide 1-tert-butyldiphenylsilyloxy-12-azidododec-7(Z)-ene (1.90 g,4.10 mmol) in THF (12 mL) containing 4 drops of deionized water. After12 h, the reaction mixture was diluted with CH₂Cl₂ (10 mL), dried, andconcentrated in vacuo to give the title compound (1.36 g, 76%) as aviscous, colorless oil that was used directly in the next reactionwithout further purification. TLC: MeOH/CH₂Cl₂ (1:4), R_(f)˜0.25; ¹H NMR(400 MHz) δ 7.62-7.68 (m, 4H), 7.32-7.40 (m, 6H), 5.30-5.40 (m, 2H),3.63 (t, J=5.2 Hz, 2H), 2.62 (t, J=4.8 Hz, 2H), 1.92-2.06 (m, 4H),1.40-1.58 (m, 4H), 1.20-1.40 (m, 81H), 1.03 (s, 9H); ¹³C NMR (100 MHz) δ135.79, 134.37, 130.42, 129.70, 127.78, 64.19, 42.28, 33.44, 32.77,29.93, 29.28, 27.40, 27.21, 27.10, 25.92, 19.44. HRMS calcd forC₂₈H₄₄NOSi [M+1]⁺438.3192. found 438.3186.

1-(12-(tert-Butyldiphenylsilyloxy)dodec-5(Z)-enyl)-3-n-pentylurea

A solution of 1-tert-butyldiphenylsilyloxy-12-aminododec-7(Z)-ene (1.32g, 3.0 mmol) in THF (5 mL) was added dropwise to a stirring solution ofn-pentyl isocyanate (0.386 mL, 3.0 mmol) in THF (10 mL). After 3 hstirring at room temperature, all volatiles were removed under reducedpressure and the residue was purified by SiO₂ column chromatographyeluting with 20% EtOAc/hexane to afford the title compound (1.26 g, 76%)as a viscous oil. TLC: EtOAc/hexanes (2:3), R_(f)˜0.40; ¹H NMR (300 MHz)δ 7.60-7.70 (m, 4H), 7.35-7.42 (m, 6H), 5.28-5.42 (m, 2H), 5.16 (br s,—NH, 2H), 3.65 (t, J=6.5 Hz, 2H), 3.08-3.20 (m, 4H), 1.96-2.08 (m, 4H),1.22-1.60 (m, 18H), 1.02 (s, 9H), 0.89 (t, J=7.3 Hz, 3H); ¹³C NMR (100MHz) δ 159.23, 135.80, 134.24, 130.52, 129.74, 129.49, 127.82, 64.22,40.62, 40.54, 32.80, 30.33, 29.95, 29.37, 29.32, 27.46, 27.34, 27.18,27.11, 25.97, 22.71, 19.46, 14.29. HRMS calcd for C₃₄H₅₅N₂O₂Si [M+1]⁺551.4033. found 551.4032.

1-(12-Hydroxydodec-5(Z)-enyl)-3-n-pentylurea

A mixture of1-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)-3-n-pentylurea (1.12g, 2.0 mmol) and tetra-n-butylammonium fluoride (2.20 mL of 1M soln inTHF, 2.2 mmol) in dry THF (10 mL) was stirred at room temperature underan argon atmosphere for 12 h, and then evaporated to dryness in vacuo.The residue was dissolved in EtOAc (50 mL) and washed with water (30mL), brine (30 mL), dried and evaporated in vacuo. Purification of theresidue via SiO₂ column chromatography gave the title compound (0.56 g,89%) as a colorless solid, mp 63.7-63.8° C. TLC: EtOAc/hexanes (7:3),R_(f)˜0.30; ¹H NMR (300 MHz) δ 5.25-5.42 (m, 2H), 4.40-4.56 (br s, —NH,2H), 3.60-3.68 (d, J=6.5 Hz, 2H), 3.08-3.20 (m, 4H), 1.96-2.14 (m, 4H),1.22-1.60 (m, 18H), 0.88 (t, J=7.0 Hz, 3H); ¹³C NMR (125 MHz) δ 159.26,130.23, 129.62, 63.72, 40.33, 40.29, 32.92, 30.30, 30.26, 29.74, 29.35,29.13, 27.26, 27.20, 27.13, 25.82, 22.69, 14.27. HRMS calcd forC₁₈H₃₇N₂O₂ [M+1]⁺ 313.2855. found 313.2857.

1-(12-Bromododec-5(Z)-enyl)-3-n-pentylurea

CBr₄ (0.55 g, 1.66 mmol) and PPh₃ (0.43 g, 1.66 mmol) were added to a 0°C. solution of 1-(12-hydroxydodec-5(Z)-enyl)-3-n-pentylurea (0.43 g,1.38 mmol) in CH₂Cl₂ (20 mL). After 2 h at room temperature, thereaction mixture was concentrated in vacuo and the residue was purifiedvia SiO₂ column chromatography to give1-(12-bromododec-5(Z)-enyl)-3-n-pentylurea (0.43 g, 83%) as a viscousoil, mp 46.7-46.8° C. TLC: EtOAc/hexanes (2:3), R_(f)˜0.60; ¹H NMR (300MHz) δ 5.22-5.42 (m, 2H), 4.40 (br s, 2H), 3.42 (t, J=9.3 Hz, 2H),3.10-3.20 (m, 4H), 1.98-2.10 (m, 4H), 1.80-1.90 (m, 2H), 1.25-1.55 (m,16H), 0.92 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 159.51, 130.14,129.69, 40.48, 40.39, 34.20, 32.96, 30.34, 29.67, 29.36, 28.58, 28.25,27.31, 27.27, 27.17, 22.68, 14.26. HRMS calcd for C₁₈H₃₆BrN₂O [M+1]⁺375.2011. found 375.2014.

1-(12-Cyanododec-5(Z)-enyl)-3-n-pentylurea

A mixture of potassium cyanide (0.23 g, 3.54 mmol) and1-(12-bromododec-5(Z)-enyl)-3-n-pentylurea (0.90 g, 2.40 mmol) wasstirred in DMSO (5 mL) at room temperature. After 12 h, the reactionmixture was diluted with water (20 mL) and extracted with ethyl acetate(2×50 mL). The combined organic extracts were washed with water (2×25mL), brine (25 mL), dried (Na₂SO₄) and was passed through a silica gelcolumn to give the title compound (0.62 g, 81%) as a colorless solid, mp56-57° C. TLC: EtOAc/hexanes (2:3), R_(f)˜0.45. ¹H NMR (300 MHz) δ5.29-5.40 (m, 2H), 4.27 (br s, —NH, 2H), 3.10-3.20 (m, 4H), 2.34 (t,J=7.0 Hz, 2H), 1.98-2.08 (m, 4H) 1.24-1.70 (m, 18H), 0.89 (t, J=7.0 Hz,3H); ¹³C NMR (125 MHz) δ 159.41, 129.94, 129.86, 120.14, 40.45, 40.35,30.30, 29.50, 29.33, 28.70, 28.51, 27.26, 27.16, 25.47, 22.66, 17.28,14.24; IR (neat) 2930, 2281, 2184, 2042, 1936, 1613, 1197, 1042 cm⁻¹.HRMS calcd for C₁₉H₃₆N₃O [M+1]⁺ 322.2858. found 322.2867.

N′-Hydroxy-13-(3-n-pentylureido)tridec-8(Z)-enimidamide

To a suspension of 1-(12-cyanododec-5(Z)-enyl)-3-n-pentylurea (350 mg,1.09 mmol) in MeOH/H₂O (4:1; 12 mL) was added H₂NOH.HCl (228 mg, 3.28mmol) and Na₂CO₃ (344 mg, 3.25 mmol). The reaction mixture was heated at60° C. for 18 h, then cooled to room temperature and all volatiles wereremoved in vacuo. The residue was diluted with water (30 mL) andextracted into ethyl acetate (2×25 mL). The combined organic extractswere washed with water (2×10 mL), brine (10 mL), dried and purified viaflash silica gel column chromatography using 5% MeOH/CH₂Cl₂ to give thetitle compound (239 mg, 62%) as a colorless solid, mp 94.6-94.7° C. TLC:MeOH/CH₂Cl₂ (1:4), R_(f)˜0.20; ¹H NMR (CD₃OD, 300 MHz) δ 5.34-5.42 (m,2H), 3.33 (s, 2H), 3.08-3.16 (m, 3H), 2.02-2.10 (m, 6H), 1.52-1.60 (m,2H), 1.44-1.52 (m, 5H), 1.30-1.44 (m, 10H), 0.92 (t, J=7.2 Hz, 3H); ¹³CNMR (CD₃OD, 125 MHz) δ 160.14, 155.23, 129.95, 129.42, 39.86, 39.76,30.70, 29.95, 29.89, 29.59, 29.06, 28.91, 27.18, 26.97, 26.95, 26.78,22.39, 13.34. HRMS calcd for C₁₉H₃₉N₄O₂ [M+1]⁺ 355.3073. found 355.3078.

Analog 25

To an ice cooled solution ofN-hydroxy-13-(3-n-pentylureido)tridec-8(Z)-enimidamide (100 mg, 0.28mmol) and pyridine (45 μL, 0.56 mmol) in THF (100 mL) was added dropwisea solution of thionyl chloride (20 μL, 0.28 mmol) in CH₂Cl₂ (2 mL).After 1 h, the reaction mixture was concentrated in vacuo, diluted withwater (25 mL), and extracted with CH₂Cl₂ (2×10 mL). The combined organicextracts were washed with water and dried. The solvent was evaporated invacuo and the residue was purified by SiO₂ column chromatography using10% MeOH/CH₂Cl₂ to give 25 (80 mg, 72%) as a sticky solid. TLC:MeOH/CH₂Cl₂ (1:9), R_(f)˜0.60; ¹H NMR (CD₃OD, 300 MHz) δ 5.33-5.36 (m,2H), 3.04-3.13 (m, 4H), 2.57 (t, J=7.4 Hz, 2H), 2.00-2.10 (m, 4H),1.62-1.74 (m, 2H), 1.25-1.54 (m, 16H), 0.92 (t, J=7.2 Hz, 3H); ¹³C NMR(CD₃OD, 125 MHz) δ 160.16, 153.94, 129.83, 129.47, 39.83, 39.71, 29.89,29.82, 29.40, 29.01, 28.59, 28.52, 26.87, 26.85, 26.71, 26.30, 23.37,22.33, 13.25. HRMS calcd for C₂₀H₃₈N₄O₂S [M+1]⁺ 398.2716. found398.2720.

Synthesis of Analog 20

Analog 20

A mixture of 1-(12-cyanododec-5(Z)-enyl)-3-n-pentylurea (500 mg, 1.55mmol), sodium azide (100 mg, 1.55 mmol) and zinc bromide (335 mg, 1.48mmol) was heated at 110° C. in isopropanol/H₂O (1:3, 8 mL) whilestirring vigorously in a sealed tube. After 18 h, the mixture was cooledto room temperature and the pH was adjusted to 1 using aq. HCl (3 N, 4mL). Ethyl acetate (10 mL) was added and the stirring was continueduntil no solid was present. The organic layer was isolated and theaqueous layer extracted with EtOAc (2×25 mL). The combined organicfractions were washed with water (3×25 mL), dried and concentrated invacuo. The residue was purified by flash silica gel columnchromatography to give the analog 20 (431 mg, 76%) as a colorless solid,mp 205.6-205.8° C. TLC: 10% MeOH/CH₂Cl₂, R_(f)˜0.30; ¹H NMR (CD₃OD, 300MHz) δ 5.40-5.30 (m, 2H), 3.06-3.11 (m, 4H), 2.93 (t, J=8.0 Hz, 2H),1.98-2.10 (m, 4H), 1.70-1.82 (m, 2H), 1.24-1.50 (m, 16H), 0.90 (t, J=7.6Hz, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ 160.16, 156.81, 129.77, 129.47,39.81, 39.68, 29.88, 29.80, 29.35, 28.99, 28.69, 28.55, 27.48, 26.85,26.81, 26.68, 22.96, 22.31, 13.22. HRMS calcd for C₁₉H₃₇N₆O [M+1]⁺365.3029. found 365.3030.

Synthesis of Analog 29

Analog 29

A mixture of N′-hydroxy-13-(3-n-pentylureido)tridec-8(Z)-enimidamide(150 mg 0.42 mmol) and 1,1′-thiocarbonyl diimidazole (90%; 91 mg, 0.51mmol) in THF (5 mL) was stirred at room temperature. After 45 min, themixture was diluted with water (10 mL) and extracted with ethyl acetate(3×5 mL). The combined extracts were washed with water, dried, andconcentrated in vacuo. The residue was dissolved in dry THF (5 mL) andboron trifluoride diethyl etherate (103 μL, 0.84 mmol) was added. Afteranother 1 h, the reaction mixture was diluted with water (20 mL) andextracted with ethyl acetate (2×10 mL). The combined extracts werewashed with water, dried (Na₂SO₄), and the solvent was evaporated invacuo. The residue was purified by column chromatography to give 29 (104mg, 63%) as a colorless solid, mp 124.2-125.1° C. TLC: MeOH/CH₂Cl₂(1:9), R_(f)˜0.60; ¹H NMR (CD₃OD, 300 MHz) δ 5.30-5.40 (m, 2H),3.02-3.12 (m, 4H), 2.54 (t, J=8.0 Hz, 2H), 1.98-2.10 (m, 4H), 1.62-1.74(m, 2H), 1.24-1.52 (m, 16H), 0.90 (t, J=7.0 Hz, 3H); ¹³C NMR (CD₃OD, 75MHz) δ 181.12, 160.13, 159.12, 129.48, 128.84, 39.85, 39.73, 30.90,29.91, 29.83, 29.41, 29.03, 28.69, 28.65, 26.89, 26.86, 26.73, 26.23,22.35, 13.29; IR (neat) 2924, 1724, 1603, 1464, 1375 cm⁻¹. HRMS calcdfor C₂₀H₃₇N₄O₂S [M+1]⁺ 397.2637. found 397.2638.

Synthesis of Analog 28

Analog 28

A mixture of N′-hydroxy-13-(3-n-pentylureido)tridec-8(Z)-enimidamide(150 mg 0.42 mmol) and 1,1′-thiocarbonyl diimidazole (90%; 91 mg, 0.51mmol) in THF (5 mL) was stirred at room temperature for 45 min. Themixture was diluted with water (20 mL) and extracted with ethyl acetate(3×10 mL). The combined organic extracts were washed with water, dried,and the solvent was evaporated in vacuo. The residue was dissolved inacetonitrile (5 mL) to which was then added DBU (147 mg, 0.96 mmol).After stirring at room temperature for 1 h, the mixture was diluted withwater 10 mL), adjusted pH4 with 1N HCl, and extracted with ethyl acetate(3×10 mL). The combined extracts were washed with water, dried overNa₂SO₄, and the solvent was evaporated in vacuo. The residue waspurified by silica gel column chromatography to give 28 (101 mg, 61%) asa colorless syrup. TLC: MeOH/CH₂Cl₂ (1:9), R_(f)˜0.55; ¹H NMR (CD₃OD,300 MHz) δ 5.30-5.40 (m, 2H), 3.04-3.14 (m, 4H), 2.62 (t, J=7.7 Hz, 2H),2.00-2.10 (m, 4H), 1.62-1.74 (m, 2H), 1.22-1.54 (m, 16H), 0.91 (t, J=6.7Hz, 3H); ¹³C NMR (CD₃OD, 125 MHz) δ 188.49, 161.91, 160.15, 129.82,129.53, 39.88, 39.75, 29.92, 29.85, 29.37, 29.05, 28.63, 28.55, 26.90,26.86, 26.75, 25.91, 23.67, 22.37, 13.31. HRMS calcd for C₂₀H₃₇N₄O₂S[M+1]⁺ 397.2637. found 397.2645.

Synthesis of Analog 11

Analog 11

A solution of 1-(12-bromododec-5(Z)-enyl)-3-n-pentylurea (300 mg, 0.79mmol), sodium sulfite (352 mg, 2.8 mmol) and cyclohexene (649 mg, 7.9mmol) in ethanol (5 mL) was refluxed overnight. The volatiles wereremoved under reduced pressure and the residue was dissolved inde-ionized water. BioRad SM-2 Bio-beads (5 g; pre-washed with 0.1N NH₄OHand H₂O) were added, gently stirred for 30 min, and then collected on asintered glass funnel. The beads were washed with deionized water (2×10mL) and then EtOH (3×10 mL). Concentration of the ethanolic washesafforded 11 (235 mg, 75%) as a colorless solid, mp 133.6-133.8° C. ¹HNMR (CD₃OD, 300 MHz) δ 5.30-5.40 (m, 2H), 3.02-3.14 (m, 4H), 2.78 (t,J=8.0 Hz, 2H), 1.98-2.12 (m, 4H), 1.72-1.84 (m, 2H), 1.22-1.50 (m, 16H),0.91 (t, J=7.0 Hz, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ 160.12, 129.86,129.49, 51.46, 39.01, 38.92, 29.92, 29.32, 29.02, 28.69, 28.42, 26.93,26.62, 25.78, 24.78, 22.34, 12.02. HRMS calcd for C₁₈H₃₅N₂NaO₄S[M]⁺398.2215. found 398.2220.

Synthesis of Analog 10

Dimethyl (12-(3-n-pentylureido)dodec-7(Z)-en-1-yl)phosphonate

A solution of 1-(12-bromododec-5(Z)-enyl)-3-n-pentylurea (250 mg, 0.67mmol) and trimethyl phosphite (10 mL) in THF (10 mL) was heated underreflux. After for 48 h, all volatiles were removed in vacuo and theresidue was purified by silica gel column chromatography using 60%EtOAc/CH₂Cl₂ to give dimethyl(12-(3-n-pentylureido)dodec-7(Z)-en-1-yl)phosphonate (160 mg, 59%) as aviscous oil. TLC: EtOAc, R_(f)˜0.55; ¹H NMR (400 MHz) δ 5.30-5.40 (m,2H), 5.10 (br s, —NH, 1H), 5.02 (br s, —NH, 1H), 3.70 (s, 3H), 3.68 (s,3H), 3.06-3.14 (m, 4H), 1.97-2.20 (m, 4H), 1.63-1.78 (m, 2H), 1.20-1.60(m, 18H), 0.88 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 159.11, 130.17,129.93, 52.59, 52.56, 40.49, 30.49, 30.32, 30.28, 29.34, 29.33, 28.58,27.21, 27.15, 27.11, 25.32, 23.93, 22.66, 22.35, 22.30, 14.24. HRMScalcd for C₂₀H₄₂N₂O₄P [M+1]⁺405.2882. found 405.2883.

Analog 10

Trimethylsilyl bromide (37 μL) was added to a solution of the abovephosphonate diester (100 mg, 0.25 mmol) in dry CHCl₃ (4 mL). After 2 hat rt, the solution was concentrated and the residue was suspended inethyl acetate (5 mL). The resultant precipitate was collected anddissolved in de-ionized water. BioRad SM-2 Bio-beads (5 g; pre-washedwith 0.1N NH₄OH and H₂O) were added, gently stirred for 1 h, and thencollected on a sintered glass funnel. The beads were washed withdeionized water (2×10 mL) and then EtOH (3×10 mL). Concentration of theethanolic washes afforded disodium phosphonate 10 (68 mg, 65%). ¹H NMR(CD₃OD, 300 MHz) δ 5.30-5.42 (m, 2H), 3.18-3.24 (m, 4H), 1.97-2.20 (m,4H), 1.50-1.78 (m, 8H), 1.20-1.60 (m, 12H), 0.92 (t, J=7.2 Hz, 3H); ¹³CNMR (CD₃OD, 75 MHz) δ 159.48, 130.10, 129.25, 40.85, 40.76, 30.48,30.25, 29.38, 29.13, 29.06, 28.81, 28.67, 27.62, 26.92, 26.70, 26.64,25.80, 22.65, 22.58, 22.25, 13.02. HRMS calcd for C₁₈H₃₅N₂Na₂O₄P[M]⁺420.2130. found 420.2122.

Synthesis of Analog 16

Analog 16

Sodium methoxide (180 μL, 30% methanolic solution) was added to asolution of 1,2,4-triazole-3-thiol (101 mg, 0.99 mmol) in dry DMF (5mL). After stirring for 10 min,1-(12-bromododec-5(Z)-enyl)-3-n-pentylurea (250 mg, 0.66 mmol) wasadded. After stirring overnight, the reaction mixture was poured intoice water (100 mL) and the resultant precipitate was collected byfiltration and dried in vacuo. The crude solid was suspended indichloromethane (100 mL), stirred for 1 h and filtered to give 16 (222mg, 85%) as a colorless solid, mp 76.1-76.2° C. TLC: EtOAc, R_(f)˜0.30;¹H NMR (CD₃OD, 300 MHz) δ 8.26 (br s, 1H), 5.29-5.40 (m, 2H), 3.04-3.14(m, 6H), 1.98-2.10 (m, 4H), 1.62-1.72 (m, 2H), 1.22-1.50 (m, 16H), 0.90(t, J=7.2 Hz, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ 160.15, 157.16, 146.90,129.86, 129.44, 39.84, 39.73, 32.10, 29.91, 29.84, 29.65, 29.49, 29.02,28.60, 28.30, 26.88, 26.73, 22.34, 13.28. HRMS calcd for C₂₀H₃₈N₅OS[M+1]⁺ 396.2797. found 396.2805.

Synthesis of Analog 17

Analog 17

Ammonium molybdate (160 mg, 0.13 mmol) and hydrogen peroxide (0.6 mL,30% aq. soln) were combined at 0° C. and stirred for 15 min. An aliquotof the resultant bright yellow solution (0.15 mL) was added dropwise toa stirring, 0° C. solution of sulfide 16 (77 mg, 0.2 mmol) in ethanol(1.0 mL) resulting in a light yellow precipitate. Over the next 15 min,aliquots (0.15 mL) of the oxidizing solution were added every 5 min.After another 10 min, the reaction mixture was partitioned between H₂Oand dichloromethane (10 mL). The aqueous phase was extracted withdichloromethane (10 mL) and the combined organic extracts were washedwith brine and dried (Na₂SO₄). The residue was purified by flash SiO₂chromatography (70% EtOAc/hexanes) to provide sulfoxide 17 (43 mg, 52%)as a colorless solid, mp 88.2-88.4° C. TLC: MeOH/EtOAc (1:9),R_(f)˜0.30; ¹H NMR (CD₃OD, 300 MHz) δ 8.38 (br s, 1H), 5.26-5.36 (m,2H), 5.18 (br s, 2H), 3.04-3.26 (m, 6H), 1.92-2.08 (m, 2H), 1.70-1.84(m, 2H), 1.20-1.50 (m, 18H), 0.86 (t, J=7.2 Hz, 3H); ¹³C NMR (CD₃OD, 75MHz) δ 163.82, 160.15, 146.98, 129.73, 129.54, 52.62, 39.81, 39.71,29.90, 29.84, 29.25, 29.01, 28.57, 28.22, 26.88, 26.77, 26.73, 22.33,21.92, 13.24. HRMS calcd for C₂₀H₃₈NsO₂S [M+1]⁺ 412.2746. found412.2741.

Synthesis of Analog 18

Analog 18

Ammonium molybdate (960 mg, 0.77 mmol) and hydrogen peroxide (3.6 mL,30% aq. soln) were combined at 0° C. and stirred for 15 min. An aliquotof the bright yellow solution (0.45 mL) was added dropwise to a 0° C.solution of sulfide 16 (154 mg, 0.39 mmol) in ethanol (3.6 mL) resultingin a light yellow precipitate. Over the next 90 min, aliquots (0.5 mL)of the oxidizing solution were added every 15 min. After another 15 min,the reaction mixture was partitioned between H₂O and dichloromethane (10mL). The aqueous phase was extracted with dichloromethane (10 mL) andthe combined organic phases were washed with brine and dried (Na₂SO₄).The residue was purified by flash SiO₂ chromatography (70%EtOAc/hexanes) to provide sulfone 18 (129 mg, 78%) as a white solid, mp90.6-90.8° C. TLC: MeOH/EtOAc (1:9), R_(f)˜0.50; ¹H NMR (CD₃OD, 300 MHz)δ 8.44 (br s, 1H), 5.25-5.30 (m, 2H), 5.02 (br s, 1H), 4.90 (br s, 1H),3.35 (t, J=7.9 Hz, 2H), 3.22-3.10 (m, 4H), 1.90-2.60 (m, 4H), 1.66-1.80(m, 2H), 1.20-1.54 (m, 16H), 0.87 (t, J=7.3 Hz, 3H); ¹³C NMR (CD₃OD, 75MHz) δ 161.15, 160.13, 145.87, 129.73, 129.56, 54.26, 39.85, 39.74,29.90, 29.83, 29.21, 29.02, 28.46, 27.80, 26.89, 26.79, 26.73, 22.34,22.19, 13.30. HRMS calcd for C₂₀H₃₈N₅O₃S [M+1]⁺ 428.2695. found428.2701.

Synthesis of Analog 23

Analog 23

To a solution of N′-hydroxy-13-(3-n-pentylureido)tridec-8(Z)-enimidamide(50 mg, 0.14 mmol) in dry dioxane (3 mL) was added1,1-carbonyldiimidazole (CDI; 27 mg, 0.16 mmol) followed by1,8-diazabicycloundec-7-ene (DBU; 23 mg, 0.15 mmol). After stirring for15 min, the reaction mixture was warmed to 110° C. for 15 min, thenreturned to room temperature. The reaction mixture was diluted withwater (20 mL) and extracted with ethyl acetate (3×5 mL). The combinedorganic extracts were washed with water, brine, dried (Na₂SO₄), andconcentrated in vacuo. The residue was purified by SiO₂ columnchromatography to give 23 (36 mg, 67%) as a sticky solid. TLC:EtOAc/hexanes (4:1), R_(f)˜0.40; ¹H NMR (CD₃OD, 300 MHz) δ 5.30-5.40 (m,2H), 3.02-3.14 (m, 4H), 2.52 (t, J=7.7 Hz, 2H), 2.00-2.10 (m, 4H),1.60-1.70 (m, 2H), 1.24-1.50 (m, 16H), 0.90 (t, J=6.7 Hz, 3H); ¹³C NMR(CD₃OD, 75 MHz) δ 158.30, 157.33, 126.98, 126.68, 37.02, 36.90, 26.97,26.53, 26.19, 25.76, 25.72, 24.02, 23.89, 22.68, 21.84, 19.52, 10.43; IR(neat) 2929, 2854, 1809, 1776, 1738, 1620, 1580, 1467, 1257, 981 cm⁻¹.HRMS (ESI-neg) calcd for C₂₀H₃₅N₄O₃ [M−1]⁻ 379.2715. found 379.2731.

Synthesis of Analog 27

N¹-n-Butyl-N²-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)oxalamide

A mixture of 2-(n-butylamino)-2-oxoacetic acid² (0.40 g, 2.70 mmol), theabove 1-tert-butyldiphenylsilyloxy-12-aminododec-7(Z)-ene (1.20 g, 2.70mmol), 1-hydroxybenzotriazole (HOBt; 0.44 g, 3.30 mmol) and[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride](0.63 g,3.30 mmol) in dry DMF (5 mL) was stirred at room temperature overnight.The reaction mixture was quenched with water (30 mL) and extracted intoethyl acetate (3×20 mL). The combined organic extracts were washed withwater (2×10 mL), brine (10 mL), dried and concentrated in vacuo. Theresidue was purified by SiO₂ column chromatography to giveN¹-n-butyl-N²-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)oxalamide(1.10 g, 73%). TLC: EtOAc/hexanes (2:3), R_(f)˜0.55; ¹H NMR (400 MHz) δ8.05 (br s, —NH, 2H), 7.66-7.74 (m, 4H), 7.32-7.42 (m, 6H), 5.30-5.42(m, 2H), 3.67 (t, J=3.9 Hz, 2H), 3.31 (q, J=5.2 Hz, 4H), 1.96-2.10 (m,4H), 1.50-1.64 (m, 6H), 1.22-1.44 (m, 10H), 1.06 (s, 9H), 0.92 (t, J=7.8Hz, 3H); ¹³C NMR (100 MHz) δ 160.33, 135.80, 134.35, 130.73, 129.74,129.20, 127.83, 64.17, 39.89, 39.69, 32.79, 31.48, 29.94, 29.29, 29.07,27.46, 27.23, 27.14, 27.0, 25.96, 20.29, 19.46, 13.96. HRMS calcd forC₃₄H₅₃N₂O₃Si [M+1]⁺ 565.3826. found 565.3824.

N¹-n-Butyl-N²-(12-hydroxydodec-5(Z)-enyl)oxalamide

N¹-n-Butyl-N²-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)oxalamide(1.20 g, 2.12 mmol) was de-silylated as described above to giveN¹-n-butyl-N²-(12-hydroxydodec-5(Z)-enyl)oxalamide (0.568 g, 82%) as acolorless solid, mp 102.8-102.9° C. TLC: EtOAc/hexanes (7:3),R_(f)˜0.55; ¹H NMR (400 MHz) δ 7.69 (br s, 2H), 5.20-5.35 (m, 2H), 3.56(t, J=4.2 Hz, 2H), 3.26 (q, J=5.6 Hz, 4H), 2.17 (br s, —OH), 1.95-2.02(m, 4H), 1.44-1.56 (m, 6H), 1.20-1.40 (m, 10H), 0.87 (t, J=7.2 Hz, 3H);¹³C NMR (100 MHz) δ 160.15, 130.66, 129.21, 62.98, 39.80, 39.63, 32.93,31.39, 29.77, 29.18, 28.95, 27.26, 27.0, 26.88, 25.80, 20.18, 13.85.HRMS calcd for C₁₈H₃₅N₂O₃ [M+1]⁺ 327.2648. found 327.2648.

N¹-(12-Bromododec-5(Z)-enyl)-N²-n-butyloxalamide

N¹-n-Butyl-N²-(12-hydroxydodec-5(Z)-enyl)oxalamide (330 mg, 1.0 mmol)was brominated as described above to giveN¹-(12-bromododec-5(Z)-enyl)-N²-n-butyloxalamide (330 mg, 84%) as awhite solid, mp 46.0-46.3° C. TLC: EtOAc/hexanes (3:2), R_(f)˜0.55; ¹HNMR (400 MHz) δ 7.79 (br s, —NH, 1H), 7.77 (br s, —NH, 1H), 5.20-5.32(m, 2H), 3.32 (t, J=6.4 Hz, 2H), 3.22 (q, J=7.2 Hz, 4H), 1.90-2.00 (m,4H), 1.72-1.82 (m, 2H), 1.42-1.56 (m, 4H), 1.20-1.40 (m, 10H), 0.85 (t,J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 160.17, 160.15, 130.40, 129.34,39.77, 39.59, 34.12, 32.93, 31.40, 29.62, 29.0, 28.54, 27.25, 27.24,27.0, 26.91, 20.18, 13.85. HRMS calcd for C₁₈H₃₄BrN₂O₂[M+1]⁺ 389.1804.found 389.1809.

N¹-n-Butyl-N²-(12-cyanododec-5(Z)-enyl)oxalamide

N¹-(12-bromododec-5(Z)-enyl)-N²-n-butyloxalamide (250 mg, 0.642 mmol)was treated as described above with potassium cyanide to giveN¹-n-butyl-N²-(12-cyanododec-5(Z)-enyl)oxalamide (168 mg, 78%) as acolorless solid, mp 83.0-83.3° C. TLC: EtOAc/hexanes (3:2), R_(f)˜035;¹H NMR (400 MHz) δ 7.45 (br s, —NH, 2H), 5.30-5.40 (m, 2H), 3.34 (q,J=8.6 Hz, 4H), 2.32 (t, J=7.6 Hz, 2H), 1.98-2.08 (m, 4H), 1.30-1.68 (m,16H), 0.92 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 160.03 (2C), 130.03,129.08, 120.10, 39.88, 39.42, 31.22, 29.40, 28.82, 28.60, 28.42, 27.07,27.06, 26.82, 25.54, 20.06, 17.01, 13.80. HRMS calcd for C₁₉H₃₄N₃O₂[M+1]⁺ 336.2651. found 336.2650.

N¹-(13-Amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N²-n-butyloxalamide

Following the procedure described above, a mixture ofN¹-n-butyl-N²-(12-cyanododec-5(Z)-enyl)oxalamide, H₂NOH.HCl, and Na₂CO₃was converted toN¹-(13-amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N²-n-butyloxalamide (102mg, 62%) as a colorless solid, 116.3-116.4° C. TLC: MeOH/CH₂Cl₂ (1:4),R_(f)˜0.20; ¹H NMR (CD₃OD, 400 MHz) δ 5.28-5.40 (m, 2H), 3.24 (t, J=6.4Hz, 4H), 1.98-2.00 (m, 6H), 1.50-1.60 (m, 6H), 1.26-1.40 (m, 10H), 0.92(t, J=7.3 Hz, 3H); ¹³C NMR (CD₃OD, 100 MHz) δ 160.55 (2C), 156.31,130.05, 129.18, 39.23, 39.09, 31.18, 30.63, 29.51, 28.83, 28.69, 27.10,26.87, 26.59, 19.88, 12.88. HRMS calcd for C₁₉H₃₇N₄O₃ [M+1]⁺ 369.2866.found 369.2864.

Analog 27

Treatment ofN¹-(13-amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N²-n-butyloxalamide (100mg, 0.27 mmol) with 1,1′-thiocarbonyl diimidazole gave 27 (71 mg, 63%)as a white solid, mp 110.6-110.8° C. TLC: MeOH/CH₂Cl₂ (1:9), R_(f)˜0.55;¹H NMR (400 MHz) δ 8.90 (br s, NH, 1H), 7.52 (br s, NH, 2H), 5.28-5.40(m, 2H), 3.20-3.40 (m, 4H), 2.59 (t, J=7.5 Hz, 2H), 1.98-2.10 (m, 4H),1.21-1.70 (m, 16H), 0.92 (t, J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 160.12,160.08, 153.31, 130.62, 129.46, 39.93, 39.85, 31.35, 29.33, 28.94,28.89, 28.68, 27.02, 26.84, 26.69, 23.96, 20.23, 13.90. HRMS calcd forC₁₉H₃₅N₄O₄S [M+1]⁺ 415.2379. found 415.2372.

Synthesis of Analog 21

Analog 21

Following the procedure used to prepare 22, a mixture ofN¹-n-butyl-N²-(12-cyanododec-5(Z)-enyl)oxalamide (30 mg, 0.10 mmol),sodium azide (11 mg, 0.20 mmol) and zinc bromide (40 mg, 0.20 mmol) washeated in isopropanol/methanol/H₂O (1:1:3, 4 mL) to give tetrazole 21(25 mg, 74%) as a colorless solid, mp 113-114° C. TLC: 10% MeOH/CH₂Cl₂,R_(f)˜0.26; ¹H NMR (CD₃OD, 400 MHz) δ 5.40-5.35 (m, 2H), 3.26 (t, J=7.0Hz, 4H), 2.44 (t, J=7.0 Hz, 2H), 2.05-2.15 (m, 4H), 1.65-1.60 (m, 6H),1.40-1.30 (m, 10H), 0.94 (t, J=7.3 Hz, 3H); ¹³C NMR (CD₃OD, 100 MHz) δ160.12, 160.05, 156.80, 130.35, 129.50, 39.20, 39.08, 31.15, 29.33,28.66, 28.65, 28.53, 27.38, 26.83, 26.79, 26.55, 22.86, 19.85, 12.84.HRMS calcd for C₁₉H₃₅N₆O₂ [M+1]⁺ 379.2822. found 379.2814.

Synthesis of Analog 26

1-(tert-Butyldiphenylsilyloxy-12-Iodododec-7(Z)-ene

Triphenylphosphine (504 mg, 1.14 mmol) and imidazole (156 mg, 2.30 mmol)were added to a 0° C. solution of the above12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-en-1-ol (500 mg, 1.14 mmol) indry THF (25 mL) under an argon atmosphere. After 10 min, solid iodine(252 mg, 1.2 equiv) was added in portions. After stirring at roomtemperature for 3 h, the reaction mixture was quenched with sat. aq.sodium bisulfite solution (10 mL). After an additional 1 h, the solutionwas washed with water (2×30 mL) and concentrated under reduced pressure.The residue was purified by flash SiO₂ column chromatography using 10%EtOAc/hexanes as eluent to give the title compound (474 mg, 76%) as acolorless oil. TLC: 20% EtOAc/hexanes, R_(f)˜0.65; ¹H NMR (300 MHz) δ7.65-7.70 (m, 4H), 7.35-7.45 (m, 6H), 5.30-5.40 (m, 2H), 3.64 (t, J=6.4Hz, 2H), 3.18 (t, J=5.5 Hz, 2H), 1.95-2.10 (m, 4H), 1.85-1.90 (m, 2H),1.22-1.50 (m, 10H), 1.20 (s, 9H); ¹³C NMR (75 MHz) δ 135.87, 130.42,130.22, 130.20, 129.95, 127.89, 64.15, 38.35, 36.20, 32.50, 29.90,28.62, 28.32, 27.25, 27.20, 27.18, 26.22, 19.12. HRMS calcd forC₂₈H₄₂IOSi [M+1]⁺ 549.2050. found 549.2044.

1-(tert-Butyldiphenylsilyloxy-12-N-isopropylamino-dodec-7(Z)-ene

Isopropylamine (464 μL, 5.45 mmol) and K₂CO₃ (373 mg, 2.73 mmol) wereadded sequentially to a room temperature solution of1-(tert-butyldiphenylsilyloxy-12-iodododec-7(Z)-ene (500 mg, 0.91 mmol)in dry tetrahydrofuran (8 mL). The mixture was heated in a sealed tubeat 90° C. for 12 h, then cooled to rt, diluted with water (5 mL),filtered and the filtrate was extracted with ethyl acetate (3×10 mL).The combined organic extracts were dried, concentrated under reducedpressure, and the residue was purified by SiO₂ column chromatographyusing a gradient from 2% to 5% MeOH/CH₂Cl₂ as eluent to give the titleamine (335 mg, 77%) as a colorless oil. TLC: 5% MeOH/CH₂Cl₂, R_(f)˜0.3;¹H NMR (300 MHz) δ 7.62-7.70 (m, 4H), 7.34-7.44 (m, 6H), 5.30-5.40 (m,2H), 3.64 (t, J=6.4 Hz, 2H), 2.72-2.84 (m, 1H), 2.58 (t, J=7.0 Hz, 2H),1.94-2.08 (m, 4H), 1.20-1.60 (m, 12H), 1.05 (d, J=7.2 Hz, 6H), 1.04 (s,9H); ¹³C NMR (75 MHz) δ 135.81, 134.40, 132.0, 129.72, 127.81, 64.21,48.96, 47.75, 32.80, 30.33, 29.97, 29.31, 27.85, 27.44, 27.33, 27.11,25.95, 23.27, 19.46. HRMS calcd for C₃₁H₅₀NOSi [M+1]⁺ 480.3662. found480.3666.

N-(12-(tert-Butyldiphenylsilyloxy)dodec-5(Z)-enyl)-N-isopropyln-heptanamide

Solid [1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride](EDCI; 131 mg, 0.69 mmol) was added in portions to a roomtemperature solution of1-(tert-butyldiphenylsilyloxy-12-N-isopropylamino-dodec-7(Z)-ene (300mg, 0.63 mmol), DMAP (84 mg, 0.69 mmol), N-hydroxybenzotriazole (HOBt;93 mg, 0.69 mmol), and n-heptanoic acid (90 mg, 0.68 mmol) in dry DMF (5mL). After 12 h, the reaction mixture was diluted with water (10 mL) andextracted with ether (3×5 mL). The combined ethereal extracts werewashed with brine, dried, and evaporated in vacuo. The residue waspurified via SiO₂ column chromatography to give the title compound (281mg, 76%) as a colorless oil. TLC: EtOAc/hexanes (1:4), R_(f)˜0.65; ¹HNMR (300 MHz, 1:1 mixture of rotamers) δ 7.62-7.70 (m, 4H), 7.34-7.44(m, 6H), 5.30-5.40 (m, 2H), 4.60-4.70 and 4.00-4.05 (m, 1H for tworotamers), 3.05 (t, J=5.2 Hz, 2H), 3.02-3.19 (m, 2H), 2.20-2.40 (m, 2H),1.95-2.10 (m, 4H), 1.20-1.60 (m, 20H), 1.18 and 1.08 (d, J=7.0 Hz, 6Hfor two rotamers), 1.02 (s, 9H), 0.88 (t, J=7.2 Hz, 3H); ¹³C NMR (75MHz) δ 177.24, 173.35, 172.25, 136.16, 135.80, 135.14, 134.37, 134.34,130.97, 130.31, 129.74, 129.73, 129.54, 129.04, 127.82, 127.75, 64.21,64.18, 48.42, 45.68, 43.62, 41.19, 34.46, 34.02, 32.81, 32.79, 31.95,31.94, 31.75, 31.28, 30.0, 29.92, 29.55, 29.46, 29.31, 29.09, 27.87,27.52, 27.49, 27.45, 27.21, 27.13, 26.94, 26.88, 25.97, 25.95, 25.79,25.15, 22.81, 22.75, 21.63, 20.77, 19.46, 19.33, 14.34, 14.30. HRMScalcd for C₃₈H₆₂NO₂Si [M+1]⁺ 592.4550. found 592.4552.

N-(12-Hydroxydodec-5(Z)-enyl)-N-isopropyl n-heptanamide

Following the desilylation procedure above,N-(12-(tert-butyldiphenylsilyloxy)dodec-5(Z)-enyl)-N-isopropyln-heptanamide (275 mg, 0.464 mmol) was converted to the title alcohol(155 mg, 94%) as a syrup. TLC: 40% EtOAc/hexanes, R_(f)˜0.45; ¹H NMR(300 MHz, 45/55 mixture of rotamers) δ 5.30-5.46 (m, 2H), 4.62-4.72 and4.00-4.08 (m, 1H for two rotamers), 3.63 (t, J=5.4 Hz, 2H), 3.06-3.14(m, 2H), 2.22-2.36 (m, 2H), 1.98-2.10 (m, 4H), 1.24-1.70 (m, 20H), 1.17and 1.10 (d, J=6.8 Hz, 6H for two rotamers), 0.88 (t, J=7.2 Hz, 3H); ¹³CNMR (75 MHz) δ 173.23, 172.67, 130.83, 130.12, 129.71, 128.96, 62.69,62.64, 48.34, 45.56, 43.52, 41.07, 34.0, 33.91, 32.91, 32.88, 31.84,31.81, 31.18, 29.82, 29.74, 29.46, 29.33, 29.11, 27.78, 27.37, 27.35,27.16, 27.13, 26.84, 25.89, 25.81, 25.66, 22.69, 21.51, 20.65, 14.21.HRMS calcd for C₂₂H₄₄NO₂ [M+1]⁺354.3372. found 354.3380.

N-(12-Bromododec-5(Z)-enyl)-N-isopropyl-n-hexanamide

Following the procedure above, N-(12-hydroxydodec-5(Z)-enyl)-N-isopropyln-heptanamide (150 mg, 0.43 mmol) was transformed as described aboveinto the corresponding bromide (144 mg, 82%) as a syrup. TLC: 30%EtOAc/hexanes, R_(f)˜0.65; ¹H NMR (300 MHz, 45/55 ratio of rotamers) δ5.30-5.42 (m, 2H), 4.60-4.70 and 4.00-4.10 (m, 1H for two rotamers),3.42 (t, J=5.3 Hz, 2H), 3.02-3.20 (m, 2H), 2.20-2.38 (m, 2H), 1.80-2.10(m, 4H), 1.20-1.70 (m, 20H), 1.16 and 1.12 (d, J=7.2 Hz, 6H for tworotamers), 0.87 (t, J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 173.20, 172.64,130.61, 130.46, 129.96, 129.94, 129.24, 48.34, 45.56, 43.54, 41.09,34.20, 34.16, 34.05, 33.97, 32.98, 32.93, 32.60, 31.90, 31.88, 31.26,29.78, 29.68, 29.62, 29.51, 29.40, 28.94, 28.55, 28.25, 28.20, 27.81,27.69, 27.45, 27.30, 27.25, 27.18, 26.92, 25.87, 25.20, 22.75, 21.59,20.72, 14.72. HRMS calcd for C ₂₂H₄₃BrNO [M+1]⁺ 416.2528. found416.2523.

N-(12-Cyanododec-5(Z)-enyl)-N-isopropyl-n-hexanamide

Following the cyanide displacement procedure above,N-(12-bromododec-5(Z)-enyl)-N-isopropyl-n-hexanamide (500 mg, 1.20 mmol)gave the title nitrile (339 mg, 78%) as a syrup. TLC: EtOAc/hexanes(3:7), R_(f)˜0.40; ¹H NMR (500 MHz, 45/55 ratio of rotamers) δ 5.20-5.34(m, 2H), 4.52-4.62 and 3.90-4.02 (m, 1H for two rotamers), 3.00-3.10 (m,2H), 2.16-2.30 (m, 4H), 1.90-2.05 (m, 4H), 1.60-1.70 (m, 8H), 1.22-1.50(m, 12H), 1.18 and 1.11, (d, J=6.8 Hz, 6H for two rotamers), 0.88 (t,J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 173.22, 172.66, 130.42, 130.04,129.78, 129.35, 120.04, 119.99, 48.34, 45.58, 43.54, 41.07, 34.02,33.95, 31.89, 31.86, 31.22, 29.53, 29.49, 29.37, 28.73, 28.70, 28.57,28.53, 27.78, 27.39, 27.26, 27.18, 27.17, 26.91, 25.85, 25.70, 25.52,25.49, 22.73, 21.56, 20.70, 17.26, 14.27. HRMS calcd for C₂₃H₄₃N₂O[M+1]⁺ 363.3375. found 363.3375.

N-(13-Amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N-isopropyl-n-heptanamide

Following the procedure above, a mixture ofN-(12-cyanododec-5(Z)-enyl)-N-isopropyl-n-hexanamide, H₂NOH.HCl, andNa₂CO₃ was converted into the title compound (64%). TLC: MeOH/CH₂Cl₂(3:7), R_(f)˜0.30; ¹H NMR (500 MHz, 1:1 ratio of rotamers) δ 5.24-5.40(m, 2H), 4.62-4.68 (m, 0.5H), 4.50-4.60 (—NH, 2H), 3.96-4.40 (m, 0.5H),3.02-3.14 (m, 2H), 2.18-2.28 (m, 2H), 1.90-2.16 (m, 6H), 1.46-1.64 (m,8H), 1.20-1.36 (m, 12H), 1.15 and 1.08 (d, J=7.3 Hz, 6H for tworotamers), 0.85 (t, J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 173.22, 172.58,154.26, 154.21, 130.82, 130.13, 129.79, 129.08, 48.32, 45.50, 43.52,41.09, 36.03, 34.05, 33.97, 31.89, 31.86, 31.459, 31.25, 29.72, 29.67,29.53, 29.39, 29.21, 29.19, 29.11, 29.03, 27.83, 27.41, 27.24, 27.17,26.90, 26.84, 25.87, 25.70, 22.74, 21.58, 20.73, 14.26. HRMS calcd forC₂₃H₄₆N₃O₂ [M+1]⁺ 396.3590. found 396.3698.

Analog 26

N-(13-Amino-13-(hydroxyimino)tridec-5(Z)-enyl)-N-isopropyl-n-heptanamide(150 mg, 0.38 mmol) was treated with thionyl chloride at 0° C. asdescribed above to give 26 (133 mg, 68%) as a syrup. TLC: EtOAc/hexanes(1:1), R_(f)˜0.30; ¹H NMR (400 MHz, 35/65 ratio of rotamers) δ 5.22-5.40(m, 2H), 4.48-4.70 and 4.00-4.12 (m, 1H for two rotamers), 3.04-3.20 (m,2H), 2.50 and 2.64 (t, J=6.9 Hz, 2H for two rotamers), 2.22, 2.38 (t,J=8.0 Hz, 2H for two rotamers), 1.90-2.10 (m, 4H), 1.50-1.78 (m, 8H),1.20-1.40 (m, 12H), 1.22 and 1.12 (d, J=6.7 Hz 6H for two rotamers),0.88 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 173.82, 173.40, 153.39,153.26, 131.03, 130.02, 129.97, 129.10, 48.85, 46.04, 43.82, 41.50,34.13, 34.02, 31.85, 31.80, 31.23, 29.58, 29.32, 29.11, 29.09, 29.02,28.80, 28.17, 27.96, 27.32, 27.24, 27.19, 26.03, 26.75, 26.64, 26.10,24.02, 29.96, 22.75, 21.56, 21.53, 20.74, 20.72, 14.27, 14.25. HRMScalcd for C₂₄H₄₄N₃O₃S [M+1]⁺ 442.3103. found 442.3106.

Synthesis of Analog 19

Analog 19

N-(12-Cyanododec-5(Z)-enyl)-N-isopropyl-n-hexanamide (350 mg, 0.97 mmol)was treated with sodium azide as described above to give tetrazole 19(250 mg, 64%) as a sticky solid. TLC: EtOAc, R_(f)˜0.40; ¹H NMR (300MHz, 35/65 ratio of rotamers) δ 5.22-5.40 (m, 2H), 4.58-4.68 and4.02-4.18 (m, 1H for two rotamers), 3.10-3.24 (m, 2H), 2.98 (t, J=7.6Hz, 2H), 2.44 and 2.30 (t, J=7.3 Hz, 2H for two rotamers), 1.94-2.10 (m,4H), 1.72-1.84 (m, 2H), 1.50-1.70 (m, 4H), 1.18-1.40 (m, 14H), 1.21 and1.10 (d, J=7.2 Hz, 6H for two rotamers), 0.82-0.87 (m, 3H); ¹³C NMR (75MHz) δ 174.06, 173.80, 130.85, 130.04, 129.79, 129.08, 48.94, 46.32,43.97, 41.55, 34.11, 34.05, 31.79, 31.72, 31.12, 29.61, 29.61, 29.51,29.25, 29.05, 28.85, 28.22, 27.98, 27.89, 27.32, 27.19, 26.92, 25.97,23.70, 22.67, 21.51, 20.67, 14.02. HRMS calcd for C₂₃H₄₄N₅O [M+]⁺406.3546. found 406.3547.

Synthesis of Analog 22

1-tert-Butyldiphenylsilyloxy-13-(tetrahydro-2H-pyran-2-yloxy)tridec-8-yne

Following the procedure applied in the synthesis of analog 25,2-(hex-5-ynyloxy)tetrahydro-2H-pyran¹ (5.0 g, 27.40 mmol) was coupledwith 1-tert-butyldiphenylsilyloxy-7-bromoheptane (11.90 g, 27.40 mmol)to give1-tert-butyldiphenylsilyloxy-13-(tetrahydro-2H-pyran-2-yloxy)tridec-8-yne³(10.50 g, 72%) as a colorless syrup whose spectral data matchedliterature values.³ TLC: EtOAc/hexanes (1:4), R_(f)˜0.60; ¹H NMR (400MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 4.57 (t, J=4.3 Hz, 1H),3.78-3.86 (m, 2H), 3.65 (t, J=6.3 Hz, 2H), 3.32-3.54 (m, 2H), 2.10-2.22(m, 4H), 1.24-1.84 (m, 20H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ 135.80,134.38, 129.72, 127.81, 99.0, 80.72, 80.10, 67.30, 64.17, 62.49, 32.77,30.99, 29.34, 29.18, 29.13, 29.0, 27.11, 26.19, 25.91, 25.74, 19.87,19.45, 19.0, 18.86.

13-(tert-Butyldiphenylsilyloxy)tridec-5-yn-1-ol

Following the procedure applied in the synthesis of analog 25,1-tert-butyldiphenylsilyloxy-13-(tetrahydro-2H-pyran-2-yloxy)tridec-8-yne(10.0 g, 18.70 mmol) was deprotected with PPTS to give13-(tert-butyldiphenylsilyloxy)tridec-5-yn-1-ol (7.70 g, 91%) as acolorless syrup. TLC: EtOAc/hexanes (3:7), R_(f)˜0.43; ¹H NMR (300 MHz)δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.62 (t, J=5.6 Hz, 4H),2.06-2.22 (m, 4H), 1.50-1.64 (m, 14H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ135.82, 134.37, 129.76, 127.85, 80.93, 80.04, 64.21, 62.63, 32.77,32.09, 29.33, 29.13, 29.10, 27.14, 25.92, 25.63, 19.47, 19.0, 18.81.HRMS calcd for C₂₉H₄₃O₂Si [M+1]⁺ 451.3032. found 451.3032.

(Z)-13-(tert-Butyldiphenylsilyloxy)tridec-5-en-1-ol

Following the procedure applied in the synthesis of analog 25,13-(tert-butyldiphenylsilyloxy)tridec-5-yn-11-ol (7.50 g, 16.60 mmol)was subjected to semi-hydrogenation to give13-(tert-butyldiphenylsilyloxy)tridec-5(Z)-en-1-ol (6.90 g, 92%) as asyrup whose spectral values matched literature data.² TLC: EtOAc/hexanes(3:7), R_(f)˜0.45; ¹H NMR (400 MHz) δ 7.64-7.68 (m, 4H), 7.42-7.34 (m,6H), 5.28-5.42 (m, 2H), 3.68-3.67 (t, J=6.4 Hz, 4H), 1.98-2.12 (m, 4H),1.50-1.60 (m, 4H), 1.40-1.24 (m, 10H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ135.83, 134.40, 130.61, 129.74, 129.60, 127.83, 64.21, 63.08, 32.83,32.60, 29.94, 29.54, 27.50, 27.18, 27.14, 26.12, 26.01, 19.48.

1-tert-Butyldiphenylsilyloxy-13-azidotridec-8(Z)-ene

Following the procedure applied in the synthesis of analog 25,13-(tert-butyldiphenylsilyloxy)tridec-5(Z)-en-1-ol (7.0 g, 15.48 mmol)was converted to 1-tert-butyldiphenylsilyloxy-13-azidotridec-8(Z)-ene(5.30 g, 72%) obtained as a syrup. TLC: EtOAc/hexanes (1:9), R_(f)˜0.55;¹H NMR (400 MHz) δ 7.68-7.64 (m, 4H), 7.42-7.34 (m, 6H), 5.28-5.42 (m,2H), 3.64 (t, J=6.4 Hz, 2H), 3.26 (t, J=5.6 Hz, 2H), 1.96-2.10 (m, 4H),1.64-1.24 (m, 14H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ 135.86, 134.44,131.0, 129.77, 129.12, 127.85, 64.27, 51.63, 32.87, 29.95, 29.56, 28.70,27.54, 27.17, 27.05, 26.93, 26.05, 19.51. IR (neat) 2930, 2783, 2361,2331, 2094, 1109 cm⁻¹. HRMS calcd for C₂₉H₄₄N₃OSi [M+1]⁺ 478.3254. found478.3250.

1-tert-Butyldiphenylsilyloxy-13-aminotridec-8(Z)-ene

Following the procedure applied in the synthesis of analog 25,1-tert-butyldiphenylsilyloxy-13-azidotridec-8(Z)-ene (3.50 g, 7.32 mmol)was reduced with triphenylphosphine to give1-tert-butyldiphenylsilyloxy-13-aminotridec-8(Z)-ene (2.44 g, 74%) as acolorless oil. TLC: MeOH/CH₂Cl₂ (1:4), R_(f)˜0.25; ¹H NMR (400 MHz) δ7.62-7.68 (m, 4H), 7.32-7.40 (m, 6H), 5.30-5.40 (m, 2H), 3.63 (t, J=5.2Hz, 2H), 2.62 (br s, 2H), 1.92-2.06 (m, 4H), 1.40-1.58 (m, 4H),1.20-1.40 (m, 10H), 1.03 (s, 9H); ¹³C NMR (100 MHz) δ 135.79, 134.39,130.46, 129.70, 127.78, 64.22, 42.02, 32.80, 29.92, 29.52, 27.46, 27.23,27.10, 25.98, 19.44. HRMS calcd for C₂₉H₄₆NOSi [M+1]⁺ 452.3349. found452.3357.

1-(13-(tert-Butyldiphenylsilyloxy)tridec-S(L)-enyl)-3-n-pentylurea

Following the procedure applied in the synthesis of analog 25,1-tert-butyldiphenylsilyloxy-13-aminotridec-8(Z)-ene (2.35 g, 5.20 mmol)was reacted with n-pentyl isocyanate to give1-(13-(tert-butyldiphenylsilyloxy)tridec-5(Z)-enyl)-3-n-pentylurea (2.23g, 76%) as a syrup. TLC: EtOAc/hexanes (1:4), R_(f)˜0.65; ¹H NMR (500MHz) δ 7.62-7.70 (m, 4H), 7.32-7.44 (m, 6H), 5.28-5.44 (m, 2H), 4.37 (brs, 2H), 3.66 (t, J=4.2 Hz, 2H), 3.08-3.20 (m, 4H), 1.98-2.08 (m, 4H),1.20-1.60 (m, 20H), 1.05 (s, 9H), 0.90 (t, J=7.2 Hz, 3H); ¹³C NMR (125MHz) δ 159.22, 135.80, 134.12, 130.24, 129.88, 129.76, 127.81, 64.21,40.62, 40.54, 32.82, 30.31, 29.95, 29.56, 29.39, 27.51, 27.35, 27.20,27.13, 26.01, 22.72, 19.43, 14.30. HRMS calcd for C₃₅H₅₇N₂O₂Si [M+11]⁺565.4189. found 565.4186.

1-(13-Hydroxytridec-5(Z)-enyl)-3-n-pentylurea

Following the procedure applied in the synthesis of analog 25,1-(13-(tert-butyldiphenylsilyloxy)tridec-5(Z)-enyl)-3-n-pentylurea (2.30g, 4.07 mmol) was desilylated using TBAF to give1-(β-hydroxytridec-5(Z)-enyl)-3-n-pentylurea (1.22 g, 92%) as a whitesolid, mp 63.1-63.3° C. TLC: EtOAc/hexanes (7:3), R_(f)˜0.55; ¹H NMR(400 MHz) δ 5.24-5.38 (m, 2H), 4.74 (br s, —NH, 2H), 3.62 (t, J=5.6 Hz,2H), 3.06-3.18 (m, 4H), 1.98-2.06 (m, 4H), 1.20-1.60 (m, 20H), 0.86 (t,J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 159.31, 130.38, 129.63, 62.90, 40.51,40.46, 32.93, 30.30, 30.29, 29.79, 29.41, 29.34, 29.23, 27.30, 27.24,27.15, 25.98, 22.67, 14.25. HRMS calcd for C₁₉H₃₉N₂O₂ [M+1]⁺327.3012.found 327.3011.

1-(13-Bromotridec-5(Z)-enyl)-3-n-pentylurea

Following the procedure applied in the synthesis of analog 25,1-(β-hydroxytridec-5(Z)-enyl)-3-n-pentylurea (1.20 g, 3.68 mmol) wastransformed into 1-(13-bromotridec-5(Z)-enyl)-3-n-pentylurea (1.17 g,82%), obtained as a sticky solid. TLC: EtOAc/hexanes (2:3), R_(f)˜0.60;¹H NMR (500 MHz) δ 5.28-5.40 (m, 2H), 4.71 (br s, —NH, 2H), 3.40 (t,J=4.2 Hz, 2H), 3.10-3.20 (m, 4H), 1.98-2.06 (m, 4H), 1.82-1.88 (m, 2H),1.26-1.50 (m, 18H), 0.87 (t, J=7.2 Hz, 3H); ¹³C NMR (125 MHz) δ 159.38,130.34, 129.59, 40.55, 40.46, 34.29, 33.02, 30.34, 29.81, 29.38, 29.30,28.89, 28.35, 27.39, 27.33, 27.18, 22.71, 14.29. HRMS calcd forC₁₉H₃₇BrN₂O [M]⁺ 388.2089. found 388.2090.

1-(13-Cyanotridec-5(Z)-enyl)-3-n-pentylurea

1-(13-Bromotridec-5(Z)-enyl)-3-n-pentylurea (1.10 g, 2.82 mmol) wasreacted with potassium cyanide as described above in the synthesis ofanalog 25 to give 1-(13-cyanotridec-5(Z)-enyl)-3-n-pentylurea (0.69 g,73%) as a colorless solid, mp 44.3-44.4° C. TLC: EtOAc/hexanes (1:1),R_(f)˜0.32; ¹H NMR (500 MHz) δ 5.30-5.42 (m, 2H), 4.35 (br s, 2H),3.04-3.20 (m, 4H), 2.34 (t, J=7.6 Hz, 2H), 1.98-2.10 (m, 4H), 1.60-1.72(m, 2H), 1.24-1.56 (m, 18H), 0.89 (t, J=7.2 Hz, 3H); ¹³C NMR (125 MHz) δ159.24, 130.23, 129.68, 120.02, 40.24, 40.13, 30.27, 29.69, 29.32,29.07, 28.82, 28.78, 27.29, 27.26, 27.13, 25.49, 22.64, 17.30, 14.24.HRMS calcd for C₂₀H₃₈N₃O [M+1]⁺ 336.3015. found 336.3019.

1-(13-(1H-Tetrazol-5-yl)tridec-5(Z)-enyl)-3-n-pentylurea (22)

Following the procedure described above in the synthesis of analog 20, amixture of 1-(13-cyanotridec-5(Z)-enyl)-3-n-pentylurea, sodium azide,and zinc bromide was heated at 110° C. to give analog 22 (66%) as acolorless solid, mp 86.0-86.2° C. TLC: MeOH/CH₂Cl₂ (1:9), R_(f)˜0.30; ¹HNMR (CD₃OD, 300 MHz) δ 5.30-5.40 (m, 2H), 3.06-3.11 (m, 4H), 2.93 (t,J=8.0 Hz, 2H), 1.98-2.10 (m, 4H), 1.70-1.82 (m, 2H), 1.24-1.50 (m, 18H),0.90 (t, J=7.6 Hz, 3H); ¹³C NMR (CD₃OD, 100 MHz) δ 160.15, 156.21,129.89, 129.39, 39.84, 39.71, 29.89, 29.82, 29.53, 29.01, 29.00, 28.81,27.45, 26.90, 26.70, 22.89, 22.33, 13.25. HRMS calcd for C₂₀H₃₈N₆O [M]⁺378.3107. found 378.3111.

Synthesis of Analog 13

1-(tert-Butyldiphenylsilyloxy)-1-(tetrahydro-2H-pyran-2-yloxy)undec-6-yne

Following the procedure applied in the synthesis of analog 25,2-(hex-5-ynyloxy)tetrahydro-2H-pyran¹ was treated with n-BuLi and1-(tert-butyldiphenylsilyloxy-5-bromopentane⁴ to give the title compound(73%) as a colorless liquid. TLC: EtOAc/hexanes (1:4), R_(f)˜0.60; ¹HNMR (400 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 4.57 (t, J=4.3 Hz,1H), 3.78-3.86 (m, 2H), 3.65 (t, J=6.3 Hz, 2H), 3.32-3.54 (m, 2H),2.10-2.22 (m, 4H), 1.24-1.84 (m, 16H), 1.04 (s, 9H); ¹³C NMR (125 MHz) δ135.80, 134.32, 129.74, 127.84, 99.01, 80.54, 80.16, 67.30, 64.02,62.50, 32.34, 30.98, 29.18, 29.12, 27.32, 27.10, 26.17, 25.73, 25.32,19.87, 18.99, 18.86. HRMS calcd for C₃₁H₄₅O₃Si [M+1]⁺ 493.3138. found493.3144.

1-(tert-Butyldiphenylsilyloxy)undec-5-yn-1-ol

Following the procedure reported to prepare analog 25,1-(tert-butyldiphenylsilyloxy)-11-(tetrahydro-2H-pyran-2-yloxy)undec-6-ynewas cleaved with a catalytic amount of PPTS to give the title compound(72%) as a colorless liquid. TLC: EtOAc/hexanes (3:7), R_(f)˜0.43; ¹HNMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.62 (t, J=5.6 Hz,4H), 2.06-2.22 (m, 4H), 1.64-1.50 (m, 10H), 1.04 (s, 9H); ¹³C NMR (100MHz) δ 135.82, 134.33, 129.77, 127.84, 80.77, 80.11, 64.06, 62.69,32.35, 32.10, 29.10, 27.12, 25.60, 25.33, 18.97, 18.78. HRMS calcd forC₂₇H₃₉O₂Si [M+1]⁺ 423.2719. found 423.2718.

11-(tert-Butyldiphenylsilyloxy)undec-5(Z)-en-1-ol

11-(tert-Butyldiphenylsilyloxy)undec-5-yn-1-ol (6.50 g, 15.40 mmol) wassubjected to semi-hydrogenation as described above to give the titleolefin (6.07 g, 93%) as a colorless oil. TLC: EtOAc/hexanes (3:7),R_(f)˜0.45; ¹H NMR (300 MHz) δ 7.68-7.64 (m, 4H), 7.34-7.32 (m, 6H),5.28-5.42 (m, 2H), 3.68-3.60 (t, J=6.4 Hz, 4H), 2.08-1.96 (m, 4H),1.60-1.50 (m, 4H), 1.40-1.24 (m, 6H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ135.79, 134.36, 130.45, 129.71, 129.63, 127.79, 64.16, 63.15, 32.69,32.59, 29.94, 29.67, 27.43, 27.13, 27.08, 26.06, 25.68, 19.45. HRMScalcd for C₂₇H₄₁O₂Si [M+l]⁺ 425.2876. found 425.2874.

1-(tert-Butyldiphenylsilyloxy-11-azidoundec-6(Z)-ene

Following the protocol described above,11-(tert-butyldiphenylsilyloxy)undec-5(Z)-en-1-ol (6.0 g, 14.24 mmol)was converted into the title azide (4.60 g, 72%), a colorless liquid.TLC: EtOAc/hexanes (1:9), R_(f)˜0.55; ¹H NMR (300 MHz) δ 7.64-7.68 (m,4H), 7.34-7.42 (m, 6H), 5.28-5.42 (m, 2H), 3.65 (t, J=6.4 Hz, 2H), 3.25(t, J=7.1 Hz, 2H), 1.96-2.10 (m, 4H), 1.24-1.64 (m, 10H), 1.04 (s, 9H);¹³C NMR (100 MHz) δ 136.63, 135.85, 134.41, 130.83, 129.79, 129.22,127.88, 64.20, 51.62, 32.77, 29.73, 28.70, 27.53, 27.18, 27.05, 26.94,25.77, 19.52; IR (neat) 2931, 2857, 2094, 1589, 1110 cm⁻¹. HRMS calcdfor C₂₇H₄₀N₃OSi [M+1]⁺ 450.2940. found 450.2941.

1-Amino-11-(tert-butyldiphenylsilyloxy)undec-5(Z)-ene

1-(tert-Butyldiphenylsilyloxy-11-azidoundec-6(Z)-ene (4.30 g, 9.57 mmol)was reduced with triphenylphosphine as described above to give the titleamine (2.96 g, 74%) as a colorless oil. TLC: MeOH/CH₂Cl₂ (1:4),R_(f)˜0.25; ¹H NMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H),5.28-5.42 (m, 2H), 3.64 (t, J=6.4 Hz, 2H), 2.82 (t, J=4.8 Hz, 2H),1.96-2.10 (m, 4H), 1.52-1.64 (m, 4H), 1.30-1.42 (m, 6H), 1.04 (s, 9H);¹³C NMR (100 MHz) δ 135.82, 134.37, 132.37, 132.27, 130.70, 129.78,129.21, 128.93, 128.81, 127.86, 64.19, 40.88, 32.77, 29.89, 29.73,27.52, 27.37, 27.17, 27.12, 27.03, 25.76, 19.47. HRMS calcd forC₂₇H₄₂NOSi [M+1]⁺420.3046. found 420.3050.

1-(11-(tert-Butyldiphenylsilyloxy)undec-5(Z)-enyl)-3-n-pentylurea

76% as a colorless oil. TLC: EtOAc/hexanes (2:3), R_(f)˜0.45; ¹H NMR(300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 5.28-5.42 (m, 2H),4.13 (br s, 2H), 3.65 (t, J=6.4 Hz, 2H), 3.02-3.20 (m, 4H), 1.96-2.08(m, 4H), 1.20-1.60 (m, 16H), 1.04 (s, 9H), 0.89 (t, J=7.2 Hz, 3H); ¹³CNMR (100 MHz) δ 158.02, 135.79, 134.36, 130.06, 129.74, 127.90, 127.82,64.17, 40.42, 40.32, 32.52, 30.28, 29.71, 29.35, 27.62, 27.54, 27.16,27.11, 25.71, 22.69, 19.45, 14.28. HRMS calcd for C₃₃H₅₃N₂O₂Si [M+1]⁺537.3876. found 537.3876.

1-(11-Hydroxyundec-5(Z)-enyl)-3-n-pentylurea

94%, mp 62.2-62.5° C. TLC: EtOAc/hexanes (7:3), R_(f)˜0.55; ¹H NMR (300MHz) δ 5.28-5.42 (m, 2H), 4.37 (br s, 2H), 3.64 (t, J=6.4 Hz, 2H),3.02-3.20 (m, 4H), 1.96-2.10 (m, 4H), 1.20-1.60 (m, 16H), 0.89 (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz) δ 159.41, 130.30, 129.73, 62.77, 40.50, 40.34,32.75, 30.27, 30.10, 29.56, 29.33, 27.23, 27.15, 27.04, 25.56, 22.66,14.24. HRMS calcd for C₁₇H₃₅N₂O₂ [M+1]⁺299.2699. found 299.2705.

1-(11-Bromoundec-5(Z)-enyl)-3-n-pentylurea

84%, colorless oil. TLC: EtOAc/hexanes (2:3), R_(f)˜0.60; ¹H NMR (300MHz) δ 5.28-5.42 (m, 2H), 4.36 (br s, 2H), 3.32 (t, J=6.4 Hz, 2H),3.02-3.20 (m, 4H), 1.96-2.10 (m, 4H), 1.20-1.60 (m, 16H), 0.89 (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz) δ 159.20, 129.94, 40.60, 40.49, 34.19, 32.91,30.29, 29.35, 29.02, 28.01, 27.26, 27.16, 22.67, 14.26. HRMS calcd forC₁₇H₃₃BrN₂O [M]⁺ 360.1776. found 360.1773.

1-(11-(2-Hydroxyphenylthio)undec-5(Z)-enyl)-3-n-pentylurea (13)

To a solution of 2-mercaptophenol (100 mg, 0.79 mmol) in DMF (3 mL) wasadded K₂CO₃ (161 mg, 1.18 mmol) and1-(11-bromoundec-5(Z)-enyl)-3-n-pentylurea (0.29 g, 0.79 mmol). After 12h at rt, the solution was diluted with water (10 mL) and extracted withethyl acetate (3×5 mL). The combined organic extracts were washed withwater, brine and dried (Na₂SO₄). The residue was purified by SiO₂ columnchromatography to give the analog 13 (230 mg, 69%) as a sticky solid.TLC: EtOAc/hexanes (1:1), R_(f)˜0.32; ¹H NMR (300 MHz) δ 7.45 (dd,J=1.9, 7.6 Hz, 1H), 7.22-7.28 (m, 1H), 6.99 (dd, J=1.2, 8.2 Hz, 1H),6.88 (dt, J=1.2, 7.6 Hz, 1H), 5.28-5.42 (m, 2H), 4.26 (br s, 2H),3.02-3.20 (m, 4H), 2.69 (t, J=7.7 Hz, 2H), 1.94-2.08 (m, 4H), 1.20-1.60(m, 16H), 0.89 (t, J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 159.05, 157.10,135.60, 130.76, 130.11, 129.82, 120.78, 119.74, 115.02, 40.70, 40.60,36.50, 30.23, 29.68, 29.32, 28.38, 27.21, 27.11, 22.66, 14.27. HRMS(ESI-neg) calcd for C₂₃H₃₇N₂O₂S [M−1]⁻ 405.2576. found 405.2575.

Synthesis of Analog 14

1-(11-(2-Hydroxyphenylsulfonyl)undec-5(Z)-enyl)-3-n-pentylurea (14)

Following the procedure utilized to prepare analog 18, analog 13 wasoxidized to give 14 (60 mg, 75%) as a colorless liquid. TLC:EtOAc/hexanes (2:3), R_(f)˜0.32; ¹H NMR (300 MHz) δ 9.08 (br s, —OH),7.72 (dd, J=1.9, 7.4 Hz, 1H), 7.44 (dt, J=1.2, 7.3 Hz, 1H), 7.10 (d,J=7.9 Hz, 1H), 7.12 (t, J=6.4 Hz, 1H), 5.28-5.42 (m, 2H), 4.70-4.85 (m,2H), 3.40-3.60 (t, J=6.2 Hz, 2H), 3.20-3.40 (m, 4H), 1.90-2.10 (m, 4H),1.70-1.80 (m, 2H), 1.20-1.50 (m, 14H), 0.85 (t, J=7.2 Hz, 3H); ¹³C NMR(75 MHz) δ 159.25, 156.66, 136.23, 130.22, 129.78, 129.52, 122.78,120.22, 118.55, 55.97, 40.08, 40.62, 30.07, 30.02, 29.25, 28.82, 27.62,27.15, 27.05, 26.62, 22.61, 22.20, 14.24. HRMS (ESI-neg) calcd forC₂₃H₃₇N₂O₄S [M−1]⁻ 437.2474. found 437.2454.

Synthesis of Analog 15

1-n-Pentyl-3-(11-thiocyanatoundec-5(Z)-enyl)urea

A mixture of 1-(11-bromoundec-5(Z)-enyl)-3-n-pentylurea (191 mg, 0.53mmol) and potassium thiocyanate (154 mg, 1.58 mmol) in dry DMSO (4 mL)were stirred at rt. After 24 h, the reaction mixture was diluted withwater (10 mL) and extracted with ethyl acetate (3×5 mL). The combinedorganic extracts were washed with water, brine and dried (Na₂SO₄) andconcentrated in vacuo. The residue was purified by SiO₂ columnchromatography to give the title urea (116 mg, 65%) as a colorlesssyrup. TLC: EtOAc/hexanes (2:3), R_(f)˜0.32; ¹H NMR (300 MHz) δ5.28-5.42 (m, 2H), 4.42 (br s, 2H), 3.10-3.20 (m, 4H), 2.94 (t, J=7.0Hz, 2H), 2.00-2.10 (m, 4H), 1.70-1.80 (m, 2H), 1.20-1.56 (m, 14H), 0.88(t, J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 158.92, 130.20, 129.68, 112.84,40.67, 40.54, 34.23, 30.22, 30.03, 29.31, 29.08, 27.66, 27.19, 27.15,27.04, 22.66, 14.27; IR (neat) 2929, 2856, 2153, 1630, 1573, 1456, 1256cm⁻¹. HRMS calcd for C₁₈H₃₄N₃OS [M+1]⁺340.2423. found 340.2421.

1-(11-(1H-Tetrazol-5-ylthio)undec-5(Z)-enyl)-3-pentylurea (15)

Following the procedure utilized to prepare analog 19,1-n-pentyl-3-(11-thiocyanatoundec-5(Z)-enyl)urea (150 mg, 0.44 mmol) wastreated with sodium azide to give analog 15 (104 mg, 62%) as a stickysolid. TLC: 5% MeOH/CH₂Cl₂, R_(f)˜0.40; ¹H NMR (300 MHz) δ 5.28-5.42 (m,2H), 4.63 (br s, 2H), 3.30 (t, J=6.7 Hz, 2H), 3.17-3.23 (m, 4H),1.95-2.04 (m, 4H), 1.44-1.80 (m, 6H), 1.24-1.42 (m, 10H), 0.89 (t, J=7.2Hz, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ 160.17, 155.38, 129.65, 129.58,39.81, 39.69, 32.38, 29.87, 29.79, 29.36, 28.99, 28.96, 27.84, 26.84,26.72, 26.69, 22.31, 13.21. HRMS (ESI-neg) calcd for C₁₈H₃₃N₆OS [M−1]⁻381.2442. found 381.2348.

Synthesis of Analog 12

N-(11-(3-n-Pentylureido)undec-6(Z)-enyl)benzenesulfonamide (12)

To a solution of benzenesulfonamide (50 mg, 0.31 mmol) in THF/HMPA (4:1;5 mL) was added n-butyllithium (2.5 M soln in hexane, 125 μL, 0.31 mmol)at −78° C. under an argon atmosphere. A solution of1-(11-bromoundec-5(Z)-enyl)-3-n-pentylurea (115 mg, 0.32 mmol) in THF (2mL) was added dropwise. After 2 h at the same temperature, the reactionwas quenched with saturated aq. NH₄Cl (5 mL). The mixture was extractedwith EtOAc (3×5 mL) and the combined extracts were washed with water,brine, dried, and concentrated under reduced pressure. The residue waspurified by SiO₂ column chromatography to give analog 12 (44 mg, 32%) asa colorless solid, mp 73.5-73.6° C. TLC: 5% MeOH/CH₂Cl₂, R_(f)˜0.40; ¹HNMR (300 MHz) δ 7.84-7.90 (m, 2H), 7.46-7.60 (m, 3H), 5.48 (br s, —NH,1H), 5.24-5.38 (m, 2H), 4.70 (br s, —NH, 1H), 4.60 (br s, —NH, 1H),3.08-3.20 (m, 4H), 2.88-2.94 (q, J=6.4 Hz, 2H), 1.94-2.40 (m, 4H),1.20-1.58 (m, 16H), 0.88 (t, J=6.8 Hz, 3H); ¹³C NMR (75 MHz) δ 159.90,140.06, 132.69, 130.03, 129.98, 129.27, 127.19, 43.26, 40.80, 40.52,30.09, 29.99, 29.61, 29.28, 29.16, 27.02, 26.99, 26.96, 26.13, 22.63,14.25. HRMS calcd for C₂₃H₄₀N₃O₃S [M+1]⁺ 438.2790. found 438.2782.

Synthesis of Analog 24

1-(11-(2,4-Dioxothiazolidin-5-yl)undec-5(Z)-enyl)-3-n-pentylurea (24)

n-Butyllithium (1.10 mL, 2.76 mmol, 2.5 M solution in hexanes) was addeddropwise to a −78° C. solution of thiazolidine-2,4-dione (0.16 g, 1.38mmol) in dry THF/HMPA (50 mL, 4:1) under an argon atmosphere. After 30min, the reaction mixture was warmed to 0° C. over 1 h, kept at thattemperature for 2 h, and then re-cooled to −78° C. Following theaddition of a solution of 1-(11-bromoundec-5(Z)-enyl)-3-n-pentylurea(0.50 g, 1.38 mmol) in THF (15 mL), the reaction temperature was slowlyincreased to rt over 3 h and stirred further for 12 h. The reactionmixture was quenched with sat. aq. NH₄Cl (5 mL), the pH was adjusted to4 using 1M oxalic acid, and the reaction mixture was extracted withEtOAc (3×125 mL). The combined extracts were washed with water (2×100mL), brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. Theresidue was purified by SiO₂ column chromatography using 5% MeOH/CH₂Cl₂to afford analog 24 (169 mg, 31%) as a colorless solid, mp 92.8-93° C.TLC: 10% MeOH/CH₂Cl₂, R_(f)˜0.20; ¹H NMR (CD₃OD, 300 MHz) δ 5.30-5.40(m, 2H), 4.42 (dd, J=3.4, 4.2 Hz, 1H), 3.04-3.13 (m, 4H), 2.00-2.16 (m,4H), 1.80-1.96 (m, 2H), 1.24-1.58 (m, 16H), 0.91 (t, J=6.7 Hz, 3H); ¹³CNMR (75 MHz) δ 177.09, 172.56, 160.15, 129.68, 129.60, 51.89, 39.69,32.57, 29.90, 29.83, 29.23, 29.02, 28.43, 26.88, 26.73, 26.47, 22.35,13.27. HRMS calcd for C₂₀H₃₆N₃O₃S [M+1]⁺ 398.2477. found 398.2477.

Synthesis of Analog 7

(S)-Dimethyl 2-(13-(3-n-pentylureido)tridec-8(Z)-enamido)succinate

L-Aspartic acid dimethyl ester hydrochloride (38 mg, 0.19 mmol) and HATU(67 mg, 0.18 mmol) were added to a stirring solution of13-(3-n-pentylureido)tridec-8(Z)-enoic acid² (50 mg, 0.15 mmol) inanhydrous DMF (20 mL) under an argon atmosphere. After 5 min,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI; 33mg, 0.17 mmol) was added followed by diisopropylethylamine (33 μL, 0.19mmol). After 12 h, the reaction mixture was diluted with EtOAc (30 mL),washed with water (30 mL), and brine (20 mL). The combined aqueouslayers were back-extracted with EtOAc (3×30 mL). The combined organicextracts were dried over Na₂SO₄, concentrated under reduced pressure,and the residue was purified by SiO₂ column chromatography using 50%EtOAc/hexanes as eluent to give the title diester (60 mg, 84%) as aviscous oil. TLC: EtOAc/hexanes (3:2), R_(f)˜0.30; ¹H NMR (300 MHz) δ6.62 (d, J=7.0 Hz, 1H), 5.22-5.40 (m, 2H), 4.85-5.04 (m, 1H), 4.80-4.88(m, 2H), 3.75 (s, 3H), 3.66 (s, 3H), 3.10-3.20 (m, 4H), 3.01 (dd, J=4.3,10 Hz, 1H), 2.82 (dd, J=4.6, 10 Hz, 1H), 2.25 (t, J=8.3 Hz, 2H),1.98-2.07 (m, 4H), 1.60-1.68 (m, 4H), 1.20-1.50 (m, 14H), 0.88 (t, J=6.7Hz, 3H); ¹³C NMR (75 MHz) δ 173.51, 171.81, 171.52, 159.02, 130.33,129.76, 53.05, 52.30, 48.57, 40.61, 40.55, 36.61, 36.28, 30.24, 30.20,29.51, 29.31, 29.10, 28.88, 27.20, 27.12, 25.72, 22.66, 14.26.

(S)-2-(13-(3-n-Pentylureido)tridec-8(Z)-enamido)succinic acid (7)

LiOH (2 mL, 2 M aqueous solution) was added to a 0° C. solution of theabove (S)-dimethyl 2-(13-(3-n-pentylureido)tridec-8(Z)-enamido)succinate(60 mg, 0.12 mmol) in THF (25 mL) and deionized H₂O (4 mL). Afterstirring at room temperature overnight, the reaction mixture was cooledto 0° C., the pH was adjusted to 4 with 1M aq. oxalic acid, and themixture was extracted with EtOAc (3×15 mL). The combined extracts werewashed with water (30 mL), brine (25 mL), dried over anhydrous Na₂SO₄,and concentrated in vacuo. The residue was purified by SiO₂ columnchromatography using 25% EtOAc/hexanes as eluent to give analog 7 (48mg, 85%) as a colorless oil. TLC: 5% MeOH/EtOAc (3:2), R_(f)˜0.30; ¹HNMR (CD₃OD, 300 MHz) δ 5.30-5.38 (m, 2H), 4.72 (t, J=4.2 Hz, 1H),3.26-3.32 (m, 4H), 2.86 (dd, J=4.3, 10 Hz, 1H), 2.77 (dd, J=4.6, 10 Hz,1H), 2.22 (t, J=7.7 Hz, 2H), 1.98-2.10 (m, 4H), 1.54-1.64 (m, 4H),1.20-1.52 (m, 14H), 0.89 (t, J=6.7 Hz, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ174.93, 173.01, 172.83, 160.17, 129.94, 129.39, 49.0, 39.81, 39.73,35.78, 35.60, 29.90, 29.82, 29.49, 29.02, 28.87, 26.90, 26.73, 25.70,22.35, 13.29. HRMS calcd for C₂₃H₄₂N₃O₆ [M+1]⁺ 456.3074. found 456.3071.

Synthesis of Analog 3

2-(2-(2-Hydroxyethoxy)ethoxy)ethyl13-(2-(n-butylamino)-2oxoacetamido)tridec-8(Z)-enoate (3)

Triethylene glycol (0.12 g, 0.8 mmol; dried over molecular sieves) wasadded to a solution of13-(2-(n-butylamino)-2-oxoacetamido)tridec-8(Z)-enoic acid² (30 mg, 0.08mmol) and N,N-dimethylaminopyridine (DMAP, 11 mg, 0.09 mmol) inanhydrous dichloromethane (10 mL) under an argon atmosphere at roomtemperature. After 3 min, solid EDCI (18 mg, 0.09 mmol) was added. After12 h, the reaction mixture was diluted with EtOAc (10 mL), washed withwater (5 mL), and concentrated in vacuo. The residue was purified bySiO₂ column chromatography using EtOAc to give analog 3 (33 mg, 82%) asa colorless solid, mp 71.7-71.9° C. TLC: EtOAc/hexanes (4:1),R_(f)˜0.30; ¹H NMR (300 MHz) δ 7.46 (br s, 2H), 5.24-5.40 (m, 2H), 4.23(t, J=4.6 Hz, 2H), 3.58-3.78 (m, 10H), 3.27 (apparent q, J=6.7 Hz, 4H),2.32 (t, J=7.6 Hz, 2H), 1.50-1.66 (m, 6H), 1.24-1.44 (m, 14H), 0.92 (t,J=6.7 Hz, 3H); ¹³C NMR (100 MHz) δ 174.10, 160.09, 130.66, 129.24,72.70, 70.76, 70.55, 69.42, 39.80, 39.61, 34.35, 31.45, 29.69, 29.21,29.12, 29.02, 27.36, 27.07, 26.92, 25.04, 20.21, 13.90. HRMS calcd forC₂₅H₄₇N₂O₇ [M+1]⁺ 487.3383. found 487.3379.

Synthesis of Analog 2

2-(2-(2-Hydroxyethoxy)ethoxy)ethyl13-(3-n-pentylureido)tridec-8(Z)-enoate (2)

13-(3-n-Pentylureido)tridec-8(Z)-enoic acid² (80 mg, 0.20 mmol) wascondensed with triethylene glycol as described above to give analog 2(86 mg, 78%) as a colorless solid, mp 42.4-42.6° C. TLC: EtOAc,R_(f)˜0.20; ¹H NMR (300 MHz) δ 5.24-5.40 (m, 2H), 4.28 (br s, 2H), 4.23(dd, J=4.9, 1.0 Hz, 2H), 3.58-3.68 (m, 10H), 3.10-3.20 (m, 4H), 2.52 (brs, —OH, 1H), 2.33 (t, J=7.6 Hz, 2H), 1.90-2.10 (m, 4H), 1.44-1.64 (m,4H), 1.22-1.40 (m, 14), 0.88 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ174.22, 158.50, 130.41, 129.62, 72.76, 70.75, 70.51, 69.38, 63.47,61.94, 40.78, 40.71, 34.33, 30.13, 30.08, 29.57, 29.25, 29.09, 28.94,27.23, 27.15, 27.06, 25.03, 22.61, 14.24. HRMS calcd for C₂₅H₄₉N₂O₆[M+1]⁺ 473.3591. found 473.3588.

Synthesis of Analog 1

2-(2-(2-Hydroxyethoxy)ethoxy)ethyl13-(N-isopropylheptanamido)tridec-8(Z)-enoate (1)

13-(N-Isopropylheptanamido)tridec-8(Z)-enoic acid² (60 mg, 0.16 mmol)was condensed with triethylene glycol as described above to give analog1 (58 mg, 73%) as a viscous, colorless oil. TLC:EtOAc (4:1), R_(f)˜0.40;¹H NMR (300 MHz, 65/35 mixture of rotamers) δ 5.26-5.40 (m, 2H),4.62-4.70 (m, 0.5H), 4.20-4.26 (m, 2H), 3.98-4.08 (m, 0.5H), 3.58-3.76(m, 10H), 3.04-3.16 (m, 2H), 2.20-2.36 (m, 4H for the two rotamers),1.98-2.10 (m, 4H), 1.46-1.66 (m, 6H), 1.24-1.38 (m, 14H), 1.18 and 1.10(d, J=7.3 Hz, 6H for two rotamers), 0.87 (t, J=7.2 Hz, 3H); ¹³C NMR (75MHz) δ 174.05, 174.01, 173.10, 172.55, 130.75, 130.11, 129.85, 129.15,72.70, 70.76, 70.55, 69.40, 63.43, 61.94, 48.30, 45.52, 43.53, 41.09,34.36, 34.32, 34.10, 34.0, 31.93, 31.89, 31.28, 29.90, 29.75, 29.69,29.56, 29.43, 29.23, 29.12, 27.84, 27.48, 27.42, 27.35, 27.18, 26.93,25.88, 25.70, 25.05, 25.02, 22.76, 21.60, 20.75, 14.28. HRMS calcd forC₂₉H₅₆NO₆[M+1]⁺ 514.4108. found 514.4111.

Synthesis of Analog 8

N-Hydroxysuccinimidyl 13-(3-n-pentylureido)tridec-8(Z)-enoate

A mixture of 13-(3-n-pentylureido)tridec-8(Z)-enoic acid² (100 mg, 0.29mmol) and N-hydroxysuccinimide (37 mg, 0.31 mmol) were azeotropicallydried using anhydrous benzene (2×5 mL), then dissolved in dry CH₂Cl₂ (5mL). To this was added EDCI (67 mg, 0.35 mmol) and DMAP (38 mg, 0.31mmol) under an argon atmosphere. After 12 h at rt, the reaction mixturewas diluted with more CH₂Cl₂ (20 mL), washed with water, brine, dried(Na₂SO₄) and concentrated in vacuo. The residue was purified by SiO₂column chromatography to give the title NHS ester (110 mg, 86%) as asticky solid that was used immediately without further purification.TLC: EtOAc/hexanes (7:3), R_(f)˜0.40; ¹H NMR (400 MHz) δ 5.27-5.36 (m,2H), 4.48 (br s, 2H), 3.09-3.15 (m, 4H), 2.81 (br s, 4H), 2.58 (t, J=7.8Hz, 2H), 1.94-2.06 (m, 4H), 1.68-1.74 (m, 2H), 1.20-1.50 (m, 16H), 0.86(t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 169.40, 168.96, 158.62, 130.29,129.71, 40.75, 40.68, 31.12, 30.18, 30.14, 29.51, 29.28, 28.80, 27.21,27.19, 27.10, 25.81, 24.72, 22.62, 14.24.

13-(3-n-Pentylureido)-N-(phenylsulfonyl)tridec-8(Z)-enamide (8)

A mixture of N-hydroxysuccinimidyl13-(3-n-pentylureido)tridec-8(Z)-enoate (150 mg, 0.34 mmol) from above,benzenesulfonamide (78 mg, 0.49 mmol) and 4-dimethylaminopyridine (DMAP;50 mg, 0.40 mmol) were heated in dry hexamethylphosphoramide (HMPA; 3mL) at 80° C. for 24 h. After cooling to rt, the reaction mixture wasdiluted with water and extracted into EtOAc (3×10 mL). The combinedextracts were washed with water, brine, dried (Na₂SO₄) and concentratedin vacuo. The residue was purified by SiO₂ column chromatography to giveanalog 8 (105 mg, 65%) as a colorless solid, mp 91.4-91.6° C. TLC:EtOAc/hexanes (3:2), R_(f)˜0.30; ¹H NMR (300 MHz) δ 8.00-8.10 (dd,J=0.9, 7.3 Hz, 2H), 7.44-7.60 (m, 3H), 5.28-5.42 (m, 2H), 5.03 (br s,—NH, 1H), 4.57 (br s, —NH, 1H), 3.21 (t, J=6.8 Hz, 2H), 3.12 (t, J=6.5Hz, 2H), 2.29 (t, J=7.9 Hz, 2H), 1.98-2.10 (m, 4H), 1.18-1.60 (m, 18H),0.90 (t, J=7.2 Hz, 3H); ¹³C NMR (75 MHz) δ 172.66, 159.28, 139.67,133.57, 130.69, 129.65, 128.93, 128.40, 41.41, 40.53, 36.22, 29.89,29.61, 29.24, 28.87, 28.33, 27.69, 26.91, 26.69, 26.47, 24.70, 22.588,14.22. HRMS calcd for C₂₅H₄₂N₃O₄S [M+1]+480.2896. found 480.2899.

Synthesis of Analog 9

N-(Methylsulfonyl)-13-(3-n-pentylureido)tridec-8(Z)-enamide (9)

N-Hydroxysuccinimidyl 13-(3-n-pentylureido)tridec-8(Z)-enoate from above(150 mg, 0.34 mmol) was reacted with methanesulfonamide (48 mg, 0.50mmol) as described above to give analog 8 (102 mg, 72%) as a colorlesssolid, mp 113.5-113.6° C. TLC: EtOAc/hexanes (1:1), R_(f)˜0.30; ¹H NMR(300 MHz) δ 5.30-5.40 (m, 2H), 3.21 (s, 3H), 3.04-3.12 (m, 4H), 2.29 (t,J=7.3 Hz, 2H), 2.00-2.10 (m, 4H), 1.22-1.66 (m, 18H), 0.90 (t, J=7.0 Hz,3H); ¹³C NMR (75 MHz) δ 175.14, 161.50, 131.17, 130.78, 41.44, 41.10,41.02, 37.13, 31.22, 31.15, 30.73, 30.34, 30.07, 30.05, 28.19, 28.17,28.03, 25.80, 23.66, 14.56. HRMS calcd for C₂₀H₄₀N₃O₄S [M+1]⁺ 418.2740.found 418.2739.

Synthesis of Analog 6

Methyl 2-(13-(1,3-dimethyl-3-n-pentylureido)tridec-8(Z)-enamido)acetate

Glycine hydrochloride (32 mg, 0.29 mmol) and 1-hydroxybenzotriazole (32mg, 0.23 mmol; HOBt) were added to a solution of13-(1,3-dimethyl-3-n-pentylureido)tridec-8(Z)-enoic acid² (70 mg, 0.19mmol) and diisopropylethylamine (50 μL, 0.29 mmol) in anhydrous DMF (20mL) under an argon atmosphere. After 5 min,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (45 mg, 0.23mmol; EDCI) was added as a solid. After stirring for 12 h at roomtemperature, the reaction mixture was diluted with water (30 mL) andextracted with EtOAc (3×30 mL). The combined organic extracts were driedover Na₂SO₄, concentrated under reduced pressure, and the residue waspurified by SiO₂ column chromatography using 30% EtOAc/hexanes as eluentto give the title methyl ester (65 mg, 79%) as a viscous oil. TLC:EtOAc/hexanes (7:3), R_(f)˜0.40; ¹H NMR (300 MHz) δ 6.18 (br s, —NH,1H), 5.26-5.40 (m, 2H), 4.04 (d, J=5.2 Hz, 2H), 3.75 (s, 3H), 3.11(apparent q, J=7.6 Hz, 4H), 2.77 (s, 3H), 2.76 (s, 3H), 2.24 (t, J=7.6Hz, 2H), 1.96-2.08 (m, 4H), 1.46-1.70 (m, 6H), 1.20-1.38 (m, 12H), 0.88(t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz) δ 170.69, 170.07, 165.65, 130.36,129.38, 52.45, 50.43, 50.42, 41.23, 35.86, 30.09, 29.62, 29.26, 29.22,29.05, 27.32, 27.15, 26.67, 25.74, 25.38, 21.65, 14.48. HRMS Calcd forC₂₄H₄₆N₃O₄ [M+1]⁺ 440.3488. found 440.3485.

2-(13-(1,3-Dimethyl-3-n-pentylureido)tridec-8(Z)-enamido)acetic acid (6)

Following the ester hydrolysis conditions described above, methyl2-(13-(1,3-dimethyl-3-n-pentylureido)tridec-8(Z)-enamido)acetate wasconverted into analog 6 (87%), obtained as a colorless liquid. TLC:EtOAc/hexanes (4:1), R_(f)˜0.40; ¹H NMR (300 MHz) δ 6.39 (br s, —NH,1H), 5.24-5.40 (m, 2H), 4.03 (d, J=4.5 Hz, 2H), 3.16 (apparent q, J=5.8Hz, 4H), 2.79 (s, 3H), 2.77 (s, 3H), 2.25 (t, J=7.0 Hz, 2H), 1.90-2.10(m, 4H), 1.48-1.70 (m, 6H), 1.20-1.40 (m, 12H), 0.88 (t, J=7.2 Hz, 3H);¹³C NMR (100 MHz) δ 174.31, 171.97, 166.01, 130.44, 129.51, 50.79,50.61, 41.72, 36.86, 36.72, 36.42, 29.64, 29.18, 29.05, 27.36, 27.26,27.09, 27.04, 25.76, 22.63, 14.24. HRMS Calcd for C₂₃H₄₄N₃O₄ [M+1]⁺426.3332. found 426.3315.

Synthesis of Analog 5

Methyl 2-(13-(N-Isopropylheptanamido)tridec-8(Z)-enamido)acetate

13-(N-Isopropyl heptanamido)tridec-8(Z)-enoic acid² (100 mg, 0.26 mmol)was condensed with glycine methyl ester as described above to give thecorresponding amide (97 mg, 82%) as a colorless syrup that was useddirectly in the next step. TLC:EtOAc (2:1), R_(f)˜0.45; ¹H NMR (300 MHz,1:1 mixture of rotamers) δ 6.25 (br s, —NH, 0.5H), 6.08 (br s, —NH,0.5H), 5.24-5.42 (m, 2H), 4.60-4.72 (m, 1H), 4.05 (d, J=2.4 Hz, 2H),3.76 (s, 1.5H), 3.75 (s, 1.5H), 3.06-3.15 (m, 2H), 2.20-2.38 (m, 4H),1.90-2.10 (m, 4H), 1.40-1.68 (m, 6H), 1.24-1.38 (m, 14H), 1.19 (d, J=6.7Hz, 3H), 1.10 (d, J=6.7 Hz, 3H), 0.88 (t, J=7.1 Hz, 3H); ¹³C NMR (100MHz) δ 173.60, 173.46, 173.16, 172.60, 170.82, 130.84, 130.12, 129.88,129.16, 53.66, 52.58, 52.53, 48.35, 45.52, 43.55, 41.38, 41.15, 36.57,34.11, 34.02, 31.95, 31.92, 31.32, 29.92, 29.72, 29.63, 29.60, 29.45,29.40, 29.26, 29.21, 28.97, 27.94, 27.45, 27.41, 27.26, 27.16, 26.96,25.91, 25.80, 25.75, 22.79, 21.62, 20.78, 14.31.

2-(13-(N-Isopropylheptanamido)tridec-8(Z)-enamido)acetic acid (5)

Following the ester hydrolysis conditions described above, methyl2-(13-(N-isopropylheptanamido)tridec-8(Z)-enamido)acetate (50 mg, 0.10mmol) was hydrolyzed to give analog 5 (44 mg, 91%) obtained as acolorless liquid. TLC:EtOAc (4:1), R_(f)˜0.20; ¹H NMR (300 MHz, 65/35mixture of rotamers) δ 6.47 and 6.35 (br s, —NH, 1H for the tworotamers), 5.24-5.42 (m, 2H), 4.60-4.70 (m, 1H), 4.05 and 4.06 (d, J=2.8Hz, 2H for the two rotamers), 3.06-3.18 (m, 2H), 2.20-2.38 (m, 4H),1.90-2.10 (m, 4H), 1.50-1.68 (m, 6H), 1.24-1.38 (m, 14H), 1.20 and 1.10(d, J=7.3 Hz, 6H for the two rotamers), 0.87 (t, J=7.2 Hz, 3H); ¹³C NMR(75 MHz) δ 174.35, 174.23, 174.18, 173.64, 172.0, 171.93, 130.98,130.25, 129.75, 129.0, 48.86, 45.99, 43.77, 41.79, 41.62, 41.48, 34.05,33.95, 31.85, 31.79, 31.13, 29.62, 29.51, 29.35, 29.31, 29.23, 28.85,28.81, 27.83, 27.44, 27.34, 27.21, 27.02, 26.90, 26.03, 25.87, 25.80,25.75, 22.75, 21.54, 20.70, 14.29. HRMS calcd for C₂₅H₄₇N₂O₄ [M+1]⁺439.3536. found 439.3531.

Synthesis of Analog 31

1-(5-(tert-Butyldiphenylsilyloxy)pentyl)-3-n-pentylurea

5-(tert-Butyldiphenylsilyloxy)pentan-1-amine⁵ (3.0 g, 8.78 mmol) wasreacted with n-pentyl isocyanate (995 mg, 8.78 mmol) as described aboveto give the title urea (85%) as a colorless oil. TLC: EtOAc/hexanes(2:3), R_(f)˜0.40; ¹H NMR (300 MHz) δ 7.60-7.70 (m, 4H), 7.30-7.40 (m,6H), 4.24 (br s, —NH, 2H), 3.64 (t, J=7.9 Hz, 2H), 3.06-3.20 (m, 4H),1.20-1.60 (m, 12H), 1.03 (s, 9H), 0.89 (t, J=7.2 Hz, 3H); ¹³C NMR (75MHz) δ 159.7, 134.90, 132.47, 132.40, 129.70, 128.81, 128.71, 127.92,63.2, 40.91, 40.81, 32.42, 29.60, 29.28, 27.11, 23.30, 22.35, 19.38.HRMS calcd for C₂₇H₄₃N₂O₂Si [M+1]⁺ 455.3094. found 455.3093.

1-(5-Hydroxypentyl)-3-n-pentylurea

1-(5-(tert-Butyldiphenylsilyloxy)pentyl)-3-n-pentylurea (3.0 g, 6.60mmol) was de-silylated as described above to give the title alcohol(1.31 g, 92%) as a colorless solid, mp 81.4-81.8° C. TLC: EtOAc/hexanes(7:3), R_(f)˜0.40; ¹H NMR (CD₃OD, 300 MHz) δ 3.54 (t, J=5.8 Hz, 2H),3.06 (q, J=6.4 Hz, 4H), 1.22-1.60 (m, 12H), 0.89 (t, J=7.3 Hz, 3H); ¹³CNMR (CD₃OD, 100 MHz) δ 160.17, 61.67, 39.82, 39.77, 32.17, 30.02, 29.91,29.03, 23.04, 22.34, 13.25. HRMS calcd for C₁₁H₂₅N₂O₂ [M+1]⁺ 217.1916.found 217.1916.

1-(5-Bromopentyl)-3-n-pentylurea

Following the protocol described above,1-(5-hydroxypentyl)-3-n-pentylurea (1.30 g, 6.02 mmol) was convertedinto the corresponding bromide (1.45 g, 87%), obtained as a colorlessoil. TLC: EtOAc/hexanes (2:3), R_(f)˜0.40; ¹H NMR (300 MHz) δ 4.44 (brs, —NH, 2H), 3.40 (t, J=6.7 Hz, 2H), 3.10-3.20 (m, 4H), 1.82-1.92 (m,2H), 1.40-1.58 (m, 6H), 1.24-1.38 (m, 4H), 0.89 (t, J=7.3 Hz, 3H); ¹³CNMR (100 MHz) δ 159.46, 40.02, 33.88, 33.02, 30.29, 29.92, 29.36, 25.83,22.12, 14.02. HRMS calcd for C₁₁H₂₄BrN₂O [M+1]⁺ 279.1072. found279.1073.

N-(4-(5-(3-n-Pentylureido)pentyloxy)benzo[d]thiazol-2-yl)acetamide (31)

A mixture of 1-(5-bromopentyl)-3-n-pentylurea (100 mg, 0.37 mmol),commercial N-(4-hydroxybenzo[d]thiazol-2-yl)acetamide (100 mg, 0.48mmol), and K₂CO₃ (67 mg, 0.48 mmol) in DMF (5 mL) was heated at 60° C.After 6 h, the reaction mixture was cooled to rt, diluted with water (25mL), and extracted into EtOAc (3×10 mL). The combined organic extractswere washed with water (2×5 mL), brine (10 mL), dried (Na₂SO₄) andconcentrated in vacuo. The residue thus obtained was purified by silicagel column chromatography to give 31 (61 mg, 40%), mp 61.6-61.8° C. TLC:EtOAc/hexane (3:2), R_(f)˜0.40; ¹H NMR (300 MHz) δ 7.39 (dd, J=0.9, 7.3Hz, 1H), 7.20 (dd, J=7.8, 7.3 Hz, 1H), 6.86 (dd, J=0.9, 7.8 Hz, 1H),4.42 (br s, —NH, 2H), 4.17 (t, J=5.4 Hz, 2H), 3.19-3.30 (m, 4H), 2.33(s, 3H), 1.82-1.98 (m, 2H), 1.64-1.80 (m, 4H), 1.42-1.60 (m, 2H),1.24-1.40 (m, 4H), 0.88 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ 170.16,159.91, 158.94, 151.37, 139.10, 133.67, 124.40, 113.94, 108.68, 68.21,40.64, 39.98, 30.27, 29.30, 28.33, 26.54, 23.25, 22.64, 22.33, 14.26.HRMS (ESI-neg) calcd for C₂₀H₂₉N₄O₃S [M−1]⁻ 405.1960. found 405.1938.

Synthesis of Analog 32

N¹-n-Butyl-N²-(5-(tert-butyldiphenylsilyloxy)pentyl)oxalamide

A mixture of 2-(n-butylamino)-2-oxoacetic acid (22 mg, 0.15),5-(tert-butyldiphenylsilyloxy)pentan-1-amine⁵ (50 mg, 0.15 mmol),N,N-diisopropylethylamine (40 mg, 0.30 mmol), and2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate methanaminium (HATU, 72 mg, 0.19 mmol) in dry DMF (5mL) was stirred at rt overnight under an argon atmosphere, then quenchedwith water (2 mL). The reaction mixture was extracted with EtOAc (3×5mL). The combined organic extracts were washed with water, dried andconcentrated in vacuo. The residue was purified by flash SiO₂ columnchromatography using EtOAc/hexanes (1:3) as eluent to give the titleoxamide (65 mg, 91%) as a colorless oil. TLC: 50% EtOAc/hexane,R_(f)˜0.56; ¹H NMR (400 MHz) δ 7.55-7.70 (m, 4H), 7.60 (br s, —NH, 2H),7.35-7.40 (m, 6H), 3.65 (t, J=6.0 Hz, 2H), 3.35-3.25 (m, 4H), 1.55-1.35(m, 10H), 1.05 (s, 9H), 0.92 (t, J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ160.28, 160.25, 135.80, 134.20, 129.85, 127.90, 63.80, 40.0, 39.70,32.40, 31.50, 29.20, 27.10, 27.05, 23.40, 20.30, 19.45, 14.0. HRMS calcdfor C₂₇H₄₁N₂O₃Si [M+1]⁺ 469.2886. found 469.2892.

N¹-n-Butyl-N²-(5-hydroxypentyl)oxalamide

A mixture ofN¹-n-butyl-N²-(5-(tert-butyldiphenylsilyloxy)pentyl)oxalamide (65 mg,0.14 mmol) and n-tetrabutylammonium fluoride (0.41 mL, 1M soln, 0.42mmol) in dry THF solution was stirred at room temperature under an argonatmosphere for 12 h. All volatiles were evaporated in vacuo, the residuewas dissolved in EtOAc (30 mL) and the organic layer was washed withwater (10 mL), brine (15 mL), dried and evaporated. Purification of theresidue via SiO₂ column chromatography using EtOAc/hexanes (1:2) aseluent gave the title compound (30 mg, 92%) as a colorless solid, mp136-137° C. TLC: 50% EtOAc/hexane, R_(f)˜0.28; ¹H NMR (300 MHz) δ 7.65(br s, NH, 1H), 7.60 (br s, —NH, 1H), 3.65 (t, J=6.0 Hz, 2H), 3.35-3.25(m, 4H), 1.90 (br s —OH, 1H), 1.60-1.50 (m, 6H), 1.40-1.35 (m, 4H), 0.92(t, J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 160.25, 160.10, 62.70, 39.80,39.65, 32.40, 31.45, 29.20, 23.25, 20.20, 13.85. HRMS calcd forC₁₁H₂₃N₂O₃ [M+1]⁺ 231.1709. found 231.1701.

N¹-(5-Bromopentyl)-N²-n-butyloxalamide

A solution of carbon tetrabromide (51 mg, 0.15 mmol) in CH₂Cl₂ (5 mL)was stirred into a 0° C. solution of triphenylphosphine (48 mg, 0.18mmol) and N¹-n-butyl-N²-(5-hydroxypentyl)oxalamide (30 mg, 0.130 mmol)in dry CH₂Cl₂ (5 mL) under an argon atmosphere. After 2 h, the reactionmixture was washed with water (5 mL), brine (10 mL), dried overanhydrous Na₂SO₄, and all volatiles were removed under reduced pressure.The residue was purified by SiO₂ column chromatography usingEtOAc/hexane (1:4) to give compound the title compound (32 mg, 84%) as acolorless solid, mp 109-110° C. TLC: 50% EtOAc/hexanes, R_(f)˜0.50; ¹HNMR (300 MHz) δ 7.55 (br s, NH, 1H), 7.50 (br s, —NH, 1H), 3.40 (t,J=6.0 Hz, 2H), 3.25-3.35 (m, 4H), 1.80-1.85 (m, 2H), 1.40-1.60 (m, 6H),1.25-1.35 (m, 2H), 0.92 (t, J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 160.30,160.15, 39.65, 33.65, 32.45, 31.40, 28.60, 25.60, 20.20, 13.90. HRMScalcd for C₁₁H₂₂BrN₂O₂[M+1]⁺ 293.0865. found 293.0870.

N²-(5-(2-Acetamidobenzo[d]thiazol-4-yloxy)pentyl)-N²-n-butyloxalamide

A mixture of N¹-(5-bromopentyl)-N²-n-butyloxalamide (32 mg, 0.11 mmol),commercial N-(4-hydroxybenzo[d]thiazol-2-yl)acetamide (22 mg, 0.11mmol), and K₂CO₃ (45 mg, 0.32 mmol) in DMF (3 mL) was heated at 80° C.for 3 h, then cooled to room temperature, diluted with water (15 mL) andextracted into EtOAc (3×10 mL). The combined organic extracts werewashed with water (2×5 mL), brine (10 mL), dried (Na₂SO₄) andconcentrated in vacuo. The residue was purified by silica gel columnchromatography using EtOAc/hexanes (1:2) as eluent to give analog 32 (35mg, 76%) as a colorless solid, mp 154-155° C. TLC: 70% EtOAc/hexanes,R_(f)˜0.35; ¹H NMR (300 MHz) δ 11.05 (br s, NH, 1H), 8.30 (br s, NH,1H), 7.70 (br s, NH, 1H), 7.40 (d, J=7.4 Hz, 1H), 7.25 (dd, J=7.4, 7.8Hz, 1H), 6.85 (d, J=7.8 Hz, 1H), 4.20 (t, J=6.0 Hz, 2H), 3.50 (q, J=7.2Hz, 2H), 3.35 (q, J=7.0 Hz, 2H), 2.35 (s, 3H), 1.95-1.90 (m, 2H),1.85-1.80 (m, 2H), 1.70-1.65 (m, 2H), 160-1.55 (m, 2H), 1.40-1.35 (m,2H), 0.95 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ 169.40, 160.55, 159.90,157.85, 151.50, 139.10, 134.00, 124.75, 113.75, 108.10, 68.90, 39.80,39.30, 31.35, 28.20, 27.40, 23.70, 23.45, 20.25, 13.90. HRMS calcd forC₂₀H₂₉N₄O₄S [M+1]⁺ 421.1910. found 421.1906.

Synthesis of Analog 30

5-(tert-Butyldiphenylsilyloxy)-N-isopropylpentan-1-amine. Followingliterature precedent,² a mixture of1-(tert-butyldiphenylsilyloxy)-5-iodopentane⁶ (1.50 g, 3.32 mmol),isopropylamine (1.70 mL, 19.92 mmol) and K₂CO₃ (1.37 g, 10.03 mmol) wasreacted to give the title amine (0.92 g, 72%) as a colorless liquid.TLC: MeOH/CH₂Cl₂ (1:4), R_(f)˜0.30; ¹H NMR (300 MHz) δ 7.65-7.67 (m,4H), 7.30-7.40 (m, 6H), 3.65 (t, J=6.4 Hz, 2H), 2.70-2.82 (m, 1H), 2.55(t, J=7.3 Hz, 2H), 1.50-1.64 (m, 2H), 1.32-1.48 (m, 4H), 1.05 (d, J=5.8Hz, 3H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ 135.68, 134.21, 129.63,127.70, 63.95, 48.81, 47.64, 32.60, 30.27, 27.0, 23.76, 23.15, 19.34.HRMS calcd for C₂₄H₃₈NOSi [M+1]⁺ 384.2723. found 384.2724.

N-(5-(tert-Butyldiphenylsilyloxy)pentyl)-N-isopropylheptanamide

Following literature precedent,²5-(tert-butyldiphenylsilyloxy)-N-isopropylpentan-1-amine (0.90 g, 2.30mmol) was condensed with heptanoic acid (0.26 g, 2.0 mmol) to give thetitle amide (0.90 g, 79%) as a viscous oil. TLC: EtOAc/hexanes (3:8),R_(f)˜0.60; ¹H NMR (300 MHz, 1:1 mixture of rotamers) δ 7.65-7.67 (m,4H), 7.30-7.40 (m, 6H), 4.62-4.72 (m, 0.5H), 4.00-4.80 (m, 0.5H), 3.62(t, J=4.8 Hz, 1H), 3.68 (t, J=4.8 Hz, 1H), 3.02 (t, J=5.2 Hz, 1H), 3.16(t, J=5.2 Hz, 1H), 2.38 (t, J=5.3 Hz, 1H), 2.24 (t, J=5.3 Hz, 1H),1.50-1.68 (m, 6H), 1.26-1.44 (m, 8H), 1.18 (d, J=7.3 Hz 3H), 1,12, (d,J=7.3 Hz 3H), 1.03 (s, 4.5H), 1.04 (s, 4.5H), 0.88 (t, J=7.3 Hz, 3H);¹³C NMR (125 MHz) δ 173.38, 172.76, 135.80, 135.78, 134.36, 134.12,129.85, 129.73, 127.88, 127.82, 64.22, 63.68, 48.43, 45.62, 43.64,41.27, 34.13, 34.05, 32.61, 32.36, 31.96, 31.93, 31.51, 29.63, 29.47,27.10, 27.03, 25.94, 25.78, 24.03, 23.77, 22.81, 21.63, 20.78, 19.47,14.33, 14.28. HRMS calcd for C31HsoNO₂Si [M+1]⁺ 496.3611. found496.3615.

N-(5-Hydroxypentyl)-N-isopropylheptanamide.N-(5-(tert-Butyldiphenylsilyloxy)pentyl)-N-isopropylheptanamide (0.70 g,1.37 mmol) was de-silylated as described above to give the title alcohol(0.34 g, 96%) as a colorless solid. TLC: EtOAc/hexanes (2:3),R_(f)˜0.30; ¹H NMR (300 MHz, 53/47 mixture of rotamers) δ 4.58-4.66 and3.96-4.08 (m, 1H for the two rotamers), 3.56 and 3.70 (t, J=5.4 Hz, 2Hfor the two rotamers), 3.02-3.16 (m, 2H), 2.30 and 2.26 (t, J=6.3 Hz, 2Hfor the two rotamers), 1.50-1.64 (m, 6H), 1.22-1.40 (m, 8H), 1.13 and1.09 (d, J=7.5 Hz, 6H for the two rotamers), 0.84 (t, J=7.3 Hz, 3H); ¹³CNMR (100 MHz) δ 172.8162.66, 62.60, 48.41, 45.55, 43.56, 41.04, 34.08,34.0, 32.46, 32.43, 31.86, 31.57, 29.40, 25.87, 25.70, 23.77, 23.73,22.75, 21.57, 20.72, 14.26. HRMS calcd for C₁₅H₃₂NO₂ [M+1]⁺ 258.2433.found 258.2436.

N-(5-Bromopentyl)-N-isopropylheptanamide

N-(5-Hydroxypentyl)-N-isopropylheptanamide (0.25 g, 0.97 mmol) wasconverted to the corresponding bromide as described above to give thetitle compound (0.25 g. 82%) as a colorless oil. TLC: EtOAc/hexanes(3:7), R_(f)˜0.40; ¹H NMR (300 MHz, 55/45 mixture of rotamers) δ4.60-4.70 and 3.96-4.10 (m, 1H for two rotamers), 3.46 and 3.36 (t,J=5.8 Hz 2H for two rotamers), 3.02-3.10 (m, 2H), 2.30 and 2.22 (t,J=7.9 Hz, 2H for two rotamers), 1.80-1.97 (m, 2H), 1.40-1.70 (m, 6H),1.20-1.40 (m, 6H), 1.16 and 1.10, (d, J=7.3 Hz, 6H for two rotamers),0.86 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ 172.79, 172.39, 48.17, 45.47,43.32, 40.70, 33.89, 33.87, 33.49, 32.48, 32.30, 31.81, 31.78, 30.78,29.27, 28.74, 26.05, 25.84, 25.69, 25.54, 22.64, 21.47, 20.62, 14.17.HRMS calcd for C₁₅H₃₁BrNO [M+1]⁺ 320.1589. found 320.1588.

N-(5-(2-Acetamidobenzo[d]thiazol-4-yloxy)pentyl)-N-isopropylheptanamide(30)

N-(5-Bromopentyl)-N-isopropylheptanamide (75 mg, 0.23 mmol) wasalkylated with commercial N-(4-hydroxybenzo[d]thiazol-2-yl)acetamide (54mg, 0.26 mmol) as described above to give analog 30 (43 mg, 42%) as asticky solid. TLC: EtOAc/hexanes (1:4), R_(f)˜0.30; ¹H NMR (300 MHz,45/55 mixture of rotamers) δ 11.50 (br s, —NH, 1H), 7.37-7.42 (m, 1H),7.18-7.26 (m, 1H), 6.84-6.88 (m, 1H), 4.58-4.78 and 4.00-4.10 (m, 1H fortwo rotamers), 4.02 (t, J=6.3 Hz, 2H), 3.12 (t, J=7.3 Hz, 2H), 2.22-2.45(m, 5H), 1.82-1.92 (m, 2H), 1.44-1.70 (m, 4H), 1.20-1.40 (m, 8H),1.11-1.19 (m, 6H), 0.82-0.95 (m, 3H); ¹³C NMR (75 MHz) δ 173.94, 172.78,169.40, 169.36, 157.89, 151.62, 138.43, 138.32, 133.91, 133.87, 124.92,124.85, 114.02, 113.87, 109.66, 108.35, 69.46, 69.08, 48.53, 48.33,45.94, 43.61, 41.20, 34.26, 34.09, 31.91, 31.88, 31.39, 29.55, 29.45,29.37, 29.30, 25.88, 25.77, 25.71, 24.43, 24.16, 23.55, 22.77, 21.59,20.80, 14.28. HRMS (ESI-neg) calcd for C₂₄H₃₆N₃O₃S [M−1]⁻ 446.2477.found 446.2434.

Synthesis of Analog 33

1-(tert-Butyldiphenylsilyloxy)-8-(tetrahydro-2H-pyran-2-yloxy)oct-3-yne

4-(tert-Butyldiphenylsilyloxy)-1-butyne⁷ (5.0 g, 16.23 mmol) was coupledwith 2-(4-bromobutoxy)tetrahydro-2H-pyran⁸ (4.59 g, 19.48 mmol) asdescribed above to give the title acetylene (5.57 g, 74%) as a colorlessoil. TLC: EtOAc/hexanes (1:9), R_(f)˜0.40; ¹H NMR (300 MHz) δ 7.66-7.69(m, 4H), 7.34-7.42 (m, 6H), 4.57 (t, J=4.3 Hz, 1H), 3.78-3.86 (m, 2H),3.73-3.90 (m, 3H), 3.32-3.40 (m, 3H), 2.40-2.45 (m, 2H), 2.06-2.10 (m,2H), 1.93-2.02 (m, 2H), 1.40-1.80 (m, 6H), 1.02 (s, 9H); ¹³C NMR (100MHz) δ 137.87, 133.96, 129.70, 128.0, 99.0, 81.0, 77.41, 67.32, 63.15,62.51, 30.97, 29.20, 27.0, 26.98, 26.0, 25.74, 23.18, 19.83, 19.44,18.88.

HRMS calcd for C₂₉H₄₁O₃Si [M+1]+465.2825. found 465.2829.

8-(tert-Butyldiphenylsilyloxy)oct-5-yn-1-ol

1-(tert-Butyldiphenylsilyloxy-8-(tetrahydro-2H-pyran-2-yloxy)oct-3-yne(5.50 g, 11.84 mmol) was de-silylated as described above to give thetitle compound (3.87 g, 86%) as a colorless oil. TLC: EtOAc/hexanes(2:3), R_(f)˜0.40; ¹H NMR (300 MHz) δ 7.60-7.68 (m, 4H), 7.30-7.40 (m,6H), 3.77 (t, J=7.4 Hz, 2H), 3.60-3.72 (m, 2H), 2.40-2.48 (m, 2H),2.22-2.40 (m, 2H), 1.50-1.70 (m, 4H), 1.03 (s, 9H); ¹³C NMR (100 MHz) δ135.83, 133.96, 129.90, 127.92, 81.31, 63.16, 62.60, 32.0, 27.0, 25.43,23.17, 19.43, 18.78. HRMS calcd for C₂₄H₃₃O₂Si [M+1]⁺ 381.2250. found381.2256.

8-(tert-Butyldiphenylsilyloxy)oct-5(Z)-en-1-ol

8-(tert-Butyldiphenylsilyloxy)oct-5-yn-1-ol (5.32 g, 14.0 mmol) wassemi-hydrogenated as described above to give8-(tert-butyldiphenylsilyloxy)oct-5(Z)-en-1-ol (5.18 g, 97%) as acolorless oil whose spectral values were in agreement with literaturedata.⁹ TLC: EtOAc/hexanes (2:3), R_(f)˜0.45; ¹H NMR (300 MHz) δ7.60-7.70 (m, 4H), 7.30-7.40 (m, 6H), 5.34-5.44 (m, 2H), 3.65 (t, J=7.0Hz, 2H), 3.58-3.64 (m, 2H), 2.23 (q, J=4.2 Hz, 2H), 1.98-2.20 (m, 2H),1.30-1.60 (m, 4H), 1.03 (s, 9H); ¹³C NMR (100 MHz) δ 135.84, 134.21,131.64, 129.82, 127.87, 126.28, 63.94, 63.12, 32.54, 31.12, 27.23,27.11, 26.02.

1-(tert-Butyldiphenylsilyloxy-8-azido-oct-3(Z)-en

Following the procedure described above,8-(tert-butyldiphenylsilyloxy)-oct-5(Z)-en-1-ol (5.20 g, 13.61 mmol) wastransformed into the title azide (3.98 g, 72%), obtained as a colorlessoil. TLC: EtOAc/hexanes (1:9), R_(f)˜0.60; ¹H NMR (300 MHz) δ 7.60-7.70(m, 4H), 7.30-7.40 (m, 6H), 5.34-5.44 (m, 2H), 3.63 (t, J=7.0 Hz, 2H),3.22 (t, J=6.4 Hz, 2H), 2.31 (q, J=3.6 Hz, 2H), 1.95-2.05 (m, 2H),1.50-1.60 (m, 2H), 1.30-1.40 (m, 2H), 1.03 (s, 9H); ¹³C NMR (100 MHz) δ135.98, 135.90, 134.28, 131.24, 130.05, 129.79, 128.10, 127.87, 126.76,63.98, 51.64, 31.24, 28.74, 27.29, 27.16, 27.07, 27.0, 19.55; IR (neat)2931, 2858, 2095, 1111 cm⁻¹. HRMS calcd for C₂₄H₃₄N₃OSi [M+1]⁺408.2471.found 408.2470.

1-(8-(tert-Butyldiphenylsilyloxy)oct-5(Z)-enyl)-3-n-pentylurea

1-(tert-Butyldiphenylsilyloxy-8-azido-oct-3(Z)-en was reduced to thecorresponding amine using triphenylphosphine as described above. Thecrude amine was reacted with n-pentyl isocyanate in THF as noted aboveand the product was purified by SiO₂ column chromatography eluting with20% EtOAc/hexane to afford the title compound (1.94 g, 84%) as a viscousoil. TLC: EtOAc/hexanes (2:3), R_(f)˜0.45; ¹H NMR (300 MHz) δ 7.60-7.70(m, 4H), 7.30-7.40 (m, 6H), 5.35-5.42 (m, 2H), 4.65 (br s, —NH, 2H),3.64 (t, J=5.5 Hz, 2H), 3.06-3.18 (m, 4H), 2.24-2.34 (q, J=3.9 Hz, 2H),1.94-2.02 (q, J=3.6 Hz, 2H), 1.20-1.50 (m, 10H), 1.03 (s, 9H), 0.83 (t,J=7.3 Hz, 3H); ¹³C NMR (100 MHz) δ 159.52, 135.85, 134.21, 131.53,129.85, 127.90, 126.28, 63.93, 40.58, 40.50, 31.13, 30.43, 30.41, 29.45,27.35, 22.77, 19.47, 14.36. HRMS calcd for C₃₀H₄₇N₂O₂Si [M+1]⁺ 495.3407.found 495.3406.

1-(8-Hydroxyoct-5(Z)-enyl)-3-n-pentylurea

1-(8-(tert-Butyldiphenylsilyloxy)oct-5(Z)-enyl)-3-n-pentylurea (3.0 g,6.07 mmol) was de-silylated as described above to give the title alcohol(1.44 g, 93%) as a colorless solid, mp 57.8-57.9° C. TLC: EtOAc/hexanes(1:4), R_(f)˜0.30; ¹H NMR (300 MHz) δ 5.30-5.60 (m, 2H), 4.40 (br s,—NH, 2H), 3.63 (t, J=6.4 Hz, 2H), 3.08-3.22 (m, 4H), 2.29 (q, J=5.3 Hz,2H), 2.09 (q, J=5.2 Hz, 2H), 1.20-1.58 (m, 100H), 0.88 (t, J=7.3 Hz,3H); ¹³C NMR (100 MHz) δ 159.48, 132.35, 126.21, 62.28, 40.54, 40.15,31.12, 30.28, 29.96, 29.35, 27.02, 26.98, 22.68, 14.27. HRMS calcd forC₁₄H₂₉N₂O₂ [M+1]⁺ 257.2229. found 257.2236.

1-(8-Bromooct-5(Z)-enyl)-3-n-pentylurea

Obtained in 82% yield as a colorless oil. TLC: EtOAc/hexanes (2:3),R_(f)˜0.60; ¹H NMR (300 MHz) δ 5.30-5.58 (m, 2H), 4.70 (br s, 2H), 3.35(t, J=6.8 Hz, 2H), 3.08-3.19 (m, 4H), 2.60 (q, J=5.6 Hz, 2H), 2.05 (q,J=5.4 Hz, 2H), 1.24-1.54 (m, 10H), 0.88 (t, J=7.3 Hz, 3H); ¹³C NMR (100MHz) δ 159.54, 132.73, 126.45, 40.53, 40.33, 32.85, 30.98, 30.33, 30.30,29.36, 27.36, 27.0, 22.69, 14.28. HRMS calcd for C₁₄H₂₉BrN₂O [M+1]⁺319.1385. found 319.1392.

N-(4-(8-(3-n-Pentylureido)oct-3(Z)-enyloxy)benzo[d]thiazol-2-yl)acetamide(33)

Obtained in 40% yield as a colorless solid, mp 113.7-113.8° C. TLC:EtOAc/hexane (3:2), R_(f)˜0.40; ¹H NMR (300 MHz) δ 12.10 (br s, —NH,1H), 7.40 (dd, J=0.8, 7.8 Hz, 1H), 7.22 (dt, J=0.6, 8.9 Hz, 1H), 6.90(dd, J=0.5, 6.9 Hz, 1H), 5.40-5.50 (m, 2H), 4.60 (br s, —NH, 2H), 4.20(t, J=5.3 Hz, 2H), 3.05-3.20 (m, 4H), 2.65 (q, J=3.9 Hz, 2H), 2.29 (s,3H), 2.15 (q, J=3.9 Hz, 2H), 1.40-1.70 (m, 10H), 0.87 (t, J=7.3 Hz, 3H);¹³C NMR (100 MHz) δ 170.07, 159.21, 158.52, 151.46, 138.41, 133.84,132.26, 126.13, 124.81, 113.81, 108.29, 68.46, 40.75, 40.64, 30.20,29.30, 27.78, 26.84, 26.47, 23.42, 22.62, 14.24. HRMS calcd forC₂₃H₃₅N₄O₃S [M+1]⁺447.2430. found 447.2431.

Synthesis of Analog 4

1-(Tetrahydro-2H-pyran-2-yloxy)-100-(tert-butyldiphenylsilyloxy)dec-5-yne

Obtained in 73% yield as a colorless oil. TLC: 15% EtOAc/hexanes,R_(f)˜0.50; ¹H NMR (500 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H),4.62 (t, J=4.3 Hz, 1H), 3.78-3.92 (m, 2H), 3.68 (t, J=6.3 Hz, 2H),3.40-3.56 (m, 2H), 2.14-2.26 (m, 4H), 1.42-1.90 (m, 14H), 1.04 (s, 9H);¹³C NMR (75 MHz) δ 135.83, 132.02, 129.80, 127.88, 99.0, 80.52, 80.30,67.31, 63.74, 62.49, 32.0, 31.06, 29.21, 27.14, 26.21, 25.82, 25.78,19.89, 18.90, 18.81. HRMS calcd for C₃₁H₄₅O₃Si [M+1]⁺ 493.3138. found493.3140.

10-(tert-Butyldiphenylsilyloxy)dec-5-yn-1-ol

Obtained in 88% yield as a colorless oil whose spectral values were inagreement with literature data.¹⁰ TLC: EtOAc/hexanes (3:7), R_(f)˜0.40;¹H NMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H), 3.67 (t, J=5.3Hz, 4H), 2.06-2.22 (m, 4H), 1.50-1.64 (m, 8H), 1.04 (s, 9H); ¹³C NMR (75MHz) δ 135.82, 135.08, 134.27, 129.80, 127.87, 80.74, 80.60, 63.67,62.69, 32.09, 31.99, 27.12, 26.83, 25.78, 25.58, 25.48, 19.49, 18.79,18.76.

10-(tert-Butyldiphenylsilyloxy)dec-5(Z)-en-1-ol

Obtained in 92% yield as a colorless oil whose spectral values were inagreement with literature data.¹⁰ TLC: EtOAc/hexanes (3:7), R_(f)˜0.45;¹H NMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.42-7.25 (m, 6H), 5.30-5.40 (m,2H), 3.67 (t, J=5.3 Hz, 4H), 2.06-2.22 (m, 4H), 1.40-1.64 (m, 8H), 1.04(s, 9H); ¹³C NMR (75 MHz) δ 135.90, 135.81, 134.37, 129.92, 129.87,129.84, 127.98, 127.80, 64.09, 63.05, 32.47, 27.24, 27.22, 27.13, 26.21,26.14, 19.51.

1-(10-Azidodec-5(Z)-enyloxy)(tert-butyldiphenylsilane

Obtained in 71% yield as a colorless oil. TLC: EtOAc/hexanes (1:9),R_(f)˜0.60; ¹H NMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.25-7.42 (m, 6H),5.30-5.40 (m, 2H), 3.65 (t, J=5.3 Hz, 2H), 3.24 (t, J=4.9 Hz, 2H),2.06-2.22 (m, 4H), 1.40-1.64 (m, 8H), 1.04 (s, 9H); ¹³C NMR (75 MHz) δ137.87, 134.35, 130.69, 129.74, 127.91, 64.02, 51.62, 32.44, 28.67,27.21, 27.16, 27.08, 26.91, 26.16, 19.48; IR (neat) 2930, 2861, 2331,2324, 2096, 1106 cm⁻¹. HRMS calcd for C₂₆H₃₈N₃OSi [M+1]⁺ 436.2784. found436.2784.

1-(10-(tert-Butyldiphenylsilyloxy)dec-5(Z)-enyl)-3-n-pentylurea

Obtained in 78% yield as a colorless oil. TLC: EtOAc/hexanes (2:3),R_(f)˜0.60; ¹H NMR (300 MHz) δ 7.64-7.68 (m, 4H), 7.34-7.42 (m, 6H),5.22-5.43 (m, 2H), 4.50 (br s, —NH, 2H), 3.65 (t, J=6.2 Hz, 2H),3.10-3.40 (m, 4H), 1.96-2.06 (m, 4H), 1.20-1.60 (m, 14H), 1.03 (s, 9H),0.88 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ 159.08, 136.03, 134.02,130.03, 128.26, 126.82, 63.28, 40.67, 40.47, 32.91, 30.48, 29.28, 27.26,27.22, 26.93, 26.02, 22.64, 19.39, 14.26. HRMS calcd for C₃₂H₅₁N₂O₂Si[M+1]⁺ 523.3720. found 523.3724.

1-(10-Hydroxydec-5(Z)-enyl)-3-n-pentylurea

Obtained in 94% yield as a colorless oil. TLC: EtOAc/hexanes (7:3),R_(f)˜0.30; ¹H NMR (400 MHz) δ 5.30-5.43 (m, 2H), 4.28 (br s, 2H), 3.63(q, J=4.6 Hz, 2H), 3.10-3.20 (m, 4H), 2.00-2.10 (m, 4H), 1.24-1.64 (m,14H), 0.88 (t, J=7.3 Hz, 3H); ¹³C NMR (75 MHz) δ 158.67, 130.17, 129.88,62.71, 40.59, 40.34, 32.52, 30.23, 30.0, 29.32, 27.09, 26.96, 25.99,22.52, 14.25. HRMS calcd for C₁₆H₃₃N₂O₂ [M+1]⁺ 285.2542. found 285.2545.

2-(10-(3-n-Pentylureido)dec-5(Z)-enyloxy)acetic acid (4)

A solution of 1-(10-hydroxydec-5(Z)-enyl)-3-n-pentylurea (66 mg, 0.23mmol) and tetra-n-butylammonium sulfate (39 mg, 0.12 mmol) inbenzene/50% aq. KOH (4 mL, 1:1) was stirred at 10° C. After 15 min,tert-butyl 2-bromoacetate (136 mg, 0.70 mmol) was added to the reactionmixture and stirred at the same temperature for an additional 1 h. Thereaction mixture was then diluted with water (10 mL) and extracted intoEtOAc (2×10 mL). The combined organic extracts were washed with water,brine and dried (Na₂SO₄) and concentrated in vacuo. The residue wasdissolved in CH₂Cl₂ (4 mL), cooled to 0° C., and trifluoroacetic acid (1mL) was added dropwise. The reaction mixture was diluted with moreCH₂Cl₂ (5 mL), washed with water, brine and dried (Na₂SO₄). The residuewas purified by SiO₂ column chromatography to give analog 4 (37 mg, 47%)as a sticky solid. TLC: EtOAc, R_(f)˜0.30; ¹H NMR (400 MHz) δ 5.30-5.42(m, 2H), 4.06 (s, 2H), 3.54 (t, J=6.6 Hz, 2H), 3.07-3.15 (m, 4H),2.00-2.12 (m, 4H), 1.21-1.66 (m, 14H), 0.88 (t, J=7.3 Hz, 3H); ¹³C NMR(75 MHz) δ 174.32, 160.08, 130.33, 129.71, 71.60, 68.12, 41.11, 41.02,29.68, 29.40, 29.17, 28.96, 27.20, 27.07, 26.64, 25.96, 22.55, 14.20.HRMS calcd for C₁₈H₃₅N₂O₄ [M+1]⁺ 343.2597. found 343.2594.

Example 2 Synthesis of Sodium(S)-2-(13-(3-pentylureido)tridec-8(Z)-enamido)succinate (NIH-F=EET A orJLJ)

As set forth in FIGS. 16A and 16B, the synthesis of EET A is as follows:

7-bromoheptane-1-ol¹ (2)

Heptane-1,7-diol (36.0 g, 272 mmol; Alfa Aesar) and aq. 48% HBr (38 mL,0.9 equiv.) were heated under reflux in benzene (400 mL) with waterremoval using a Dean-Stark apparatus. After 16 h, all volatiles wereremoved in vacuo and the residue was purified by SiO₂ columnchromatography using a gradient of 10-30% EtOAc/hexanes as eluent togive 7-bromoheptan-1-ol (26.22 g, 62%) as a colorless oil. TLC: 50%EtOAc/hexanes, R_(f)≈0.40; ¹H NMR (400 MHz, CDCl₃) δ 3.61 (t, 2H, J=7.1Hz), 3.39 (t, 2H, J=6.8 Hz), 1.80-1.88 (m, 2H), 1.52-1.58 (m, 2H),1.30-1.46 (m, 6H).

2-(7-Bromoheptyloxy)tetrahydro-2H-pyran² (3)

Dihydropyran (5.20 g, 6.11 mmol) was added to a stirring 0° C. solutionof 7-bromoheptane-1-ol (2) (11.0 g, 56.7 mmol) and a catalytic amount ofPTSA in CH₂Cl₂. After stirring at rt for 12 h, the reaction mixture wasdiluted with CH₂Cl₂ (200 mL), washed with water (100 mL×2), brine (100mL×3), dried over anhydrous sodium sulphate, and evaporated. The residuewas purified by silica gel column chromatography using a gradient of10-20% ethyl acetate/hexane as eluent to give2-(7-bromoheptyloxy)tetrahydro-2H-pyran (3) (14.50 g, 92%) as acolorless oil. TLC: 10% EtOAc/hexanes, R_(f)≈0.55; ¹H NMR (400 MHz,CDCl₃) δ 4.58-4.56 (m, 1H), 3.84-3.88 (m, 1H), 3.68-3.77 (m, 1H),3.46-3.51 (m, 1H), 3.33-3.43 (m, 3H), 1.80-1.81 (m, 2H), 1.30-1.62 (m,14H).

tert-Butyl(hex-5-yn-1-yloxy)diphenylsilane³ (5)

tert-Butyldiphenylchlorosilane (3.2 mL, 12.4 mmol) was added dropwise toa stirring, 0° C. solution of 5-hexyn-1-ol (4, 1.07 g, 10.9 mmol) andanhydrous imidazole (1.84 g, 27.1 mmol) in anhydrous CH₂Cl₂ (20 mL)under an argon atmosphere. After complete addition, the reaction mixturewas stirred at room temperature for 12 hours, then quenched withsaturated aq. NH₄Cl solution (50 mL) and extracted with Et₂O (3×50 mL).The combined ethereal extracts were washed with saturated aq. NaClsolution (25 mL), dried over NaSO₄, and the solvent was removed invacuo. The residue was purified by flash chromatography (5% ethylacetate/hexane) to yield 1-tert-butyldiphenylsilyloxy-hex-5-yne (5)(3.54 g, 10.5 mmol, 96%) as a colorless oil. TLC: 5% EtOAc/hexanes,R_(f)≈0.65; ¹H NMR (400 MHz, CDCl₃) δ 7.77-7.65 (m, 4H), 7.40-7.30 (m,6H), 3.70 (t, 2H, J=5.6 Hz), 2.40-2.25 (m, 2H), 2.05 (t, 1H, J=2.8 Hz),1.90-1.70 (m, 4H), 1.18 (s, 9H).

tert-Butyldiphenyl((tetrahydro-2H-pyran-2-yloxy)tridec-5-yn-1-yl)oxy)silane(6)

n-Butyllithium (14.3 mL, 35.9 mmol, 2.5 M solution in hexanes) was addeddropwise to a −78° C. solution oftert-butyl(hex-5-ynyloxy)diphenylsilane (10 g, 29.76 mmol) in THF anddry HMPA (4:1, 200 mL) under an argon atmosphere. After 30 min, thereaction mixture was warmed to 0° C. over a period of 1 h and held therefor 2 h. The reaction mixture was re-cooled −78° C. and a THF solution(50 mL) of bromide 3 (8.20 g, 29.3 mmol) was added. The reactiontemperature was allowed to warm to rt over 3 h and was held at thistemperature for 12 h before being quenched by adding saturated aq. NH₄Clsolution (5 mL). The pH of the reaction mixture was adjusted to −4 using1M oxalic acid and extracted with EtOAc (2×250 mL). The combined organicextracts were washed sequentially with water (2×100 mL) and brine (100mL), the organic layer was dried using anhydrous Na₂SO₄ and concentratedunder vacuo. Residue was purified by silica gel column chromatographyusing 10% EtOAc/hexanes to afford 6 (12.4 g, 78%) as a colorless thickoil. ¹H NMR (CDCl₃, 400 MHz) δ 7.68-7.64 (m, 4H), 7.42-7.34 (m, 6H),4.57 (t, J=4.3 Hz, 1H), 3.86-3.78 (m, 1H), 3.65 (t, J=6.3 Hz, 3H),3.54-3.32 (m, 4H), 2.22-2.10 (m, 4H), 1.84-1.24 (m, 18H), 1.04 (s, 9H);¹³C NMR (CDCl₃, 100 MHz) δ 135.82, 135.77, 134.25, 129.72, 127.85,127.80, 127.77, 99.09, 99.05, 80.59, 80.22, 67.84, 67.74, 63.70, 62.58,62.54, 31.00, 25.73, 19.92, 19.44, 18.97, 18.77.

13-(tert-Butyldiphenylsilyloxy)tridec-8-yn-1-ol (7)

A solution of 6 (15.0 g, 0.59 mmol) and a catalytic amount of PPTS (10mg) in MeOH (20 mL) was stirred at 0° C. for 10 h, then quenched withsaturated aq. NaHCO₃ solution. Most of the methanol was evaporated invacuo. The residue was diluted with water (100 mL) and extracted withethyl acetate (100 mL×3). The combined organic extracts wereconcentrated under reduced pressure and the residue was purified bysilica gel column chromatography using 20-30% ethyl acetate/hexane aseluent to afford 7 as a colorless oil (8.80 g, 78.7%). TLC: 20%EtOAc/hexanes, R_(f)≈0.36; ¹H NMR (CDCl₃, 400 MHz) δ 7.68-7.65 (m, 4H),7.41-7.35 (m, 6H), 3.66-3.62 (m, 4H), 2.10-1.95 (m, 4H), 1.64-1.50 (m,2H), 1.48-1.20 (m, 10H), 1.04 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ135.79, 135.77, 134.26, 129.73, 127.84, 127.83, 127.80, 127.78, 80.56,80.28, 63.72, 63.22, 32.96, 31.90, 29.01, 25.78, 19.45, 18.95, 18.77.

13-(tert-Butyldiphenylsilyloxy)tridec-8(Z)-en-1-ol (8)

In a two neck round bottom flask, NaBH₄ (176 mg, 4.65 mmol) was added insmall portions to a solution of Ni(OAc)₂.4H₂O (1.16 g, 9.3 mmol) inabsolute ethanol (10 mL) under a hydrogen atmosphere (1 atm). After 15min, dry ethylenediamine (0.56 g, 9.3 mmol) was added followed after anadditional 15 min by a solution of alcohol 7 (8.0 g, 18.7 mmol) inabsolute ethanol (25 mL). The reduction was monitored by TLC untilcomplete and then diluted with ether (50 mL), passed through a small padof silica gel to remove inorganic impurities. The filtrate wasconcentrated under reduced pressure to afford 8 as a viscous, colorlessoil (7.60 g, 95%). TLC: 50% EtOAc/hexane, R_(f)≈0.42; ¹H NMR (CDCl₃, 400MHz) δ 7.68-7.65 (m, 4H), 7.41-7.35 (m, 6H), 5.40-5.30 (m, 2H),3.58-3.65 m, 4H), 1.88-2.10 (m, 4H), 1.50-1.61 (m, 4H), 1.25-1.45 (m,10H), 1.04 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 135.83, 134.36, 130.30,129.98, 129.77, 127.85, 64.09, 63.17, 32.48, 29.96, 29.53, 27.49, 27.37,27.21, 27.16, 26.23, 26.01, 19.50.

13-(tert-Butyldiphenylsilyloxy)tridec-8(Z)-enoic acid (9)

Jones reagent (5.8 mL of a 10 N solution in water) in acetone (25 mL)was added to a stirring, −40° C. solution of alcohol 8 (5.0 g, 11.8mmol) in acetone (75 mL). After 1 h, the reaction mixture was warmed to−10° C. and maintained for another 2 h, then quenched with an excess(5.0 equiv) of isopropanol. The green chromium salts were removed byfiltration and the filter cake was washed with acetone. The combinedfiltrates and washings were concentrated in vacuo and the resultantresidue was dissolved in EtOAc (100 mL), washed with water (50 mL),dried over anhydrous sodium sulphate, and concentrated in vacuo. Theresidue was purified by SiO₂ column chromatography using 15%EtOAc/hexanes as eluent to give 9 (3.84 g, 74.20%) as a liquid. TLC: 40%EtOAc/hexanes, R_(f)≈0.40. ¹H NMR (CDCl₃, 400 MHz) δ 7.68-7.64 (m, 4H),7.43-7.34 (m, 6H), 5.40-5.26 (m, 2H), 3.66 (t, J=6.6 Hz, 2H), 2.35 (t,J=7.3 Hz, 2H), 2.10-1.90 (m, 4H) 1.64-1.50 (m, 2H), 1.48-1.20 (m, 10H),1.04 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 180.62, 135.81, 134.34, 130.10,130.07, 129.73, 127.82, 127.80, 127.79, 64.06, 32.44, 29.12, 27.38,27.18, 27.12, 26.19, 24.87, 19.47.

Methyl β-hydroxytridec-8(Z)-enoate⁴ (10)

A solution of 9 (7.60 g, 3.49 mmol) and p-toluenesulphonic acid (50 mg;PTSA) in MeOH (50 mL) was stirred at room temperature for 4 h, and thenconcentrated in vacuo. The residue was purified by SiO₂ columnchromatography using 25% EtOAc/hexanes as eluent to give 10 (3.41 g,87%) as a colorless oil. TLC: 40% EtOAc/hexanes, R_(f)≈0.35; ¹H NMR(CDCl₃, 400 MHz) δ 5.40-5.36 (m, 2H), 3.60-3.66 (m, 5H), 2.30 (t, J=7.3Hz, 2H), 2.10-1.90 (m, 4H) 1.64-1.50 (m, 2H), 1.48-1.20 (m, 10H).

Methyl 13-azidotridec-8(Z)-enoate⁵ (11)

Diisopropyl azodicarboxylate (DIAD; 3.0 g, 14.8 mmol) was added dropwiseto a −20° C. solution of triphenylphosphine (3.9 g, 14.8 mmol) in dryTHF (100 mL) under an argon atmosphere. After stirring for 10 min, asolution of 10 (3.0 g, 4.75 mmol) in anhydrous THF (5 mL) was addeddropwise. After 30 min at −20° C., the reaction mixture was warmed to 0°C. and diphenylphosphorylazide (DPPA, 4.0 g, 14.5 mmol) was addeddropwise. After stirring at room temperature for 6 h, the reaction wasquenched with water (3 mL), diluted with ether (100 mL), and washed withbrine (40 mL). The aqueous layer was back-extracted with ether (2×150mL). The combined organic extracts were dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by SiO₂column chromatography using 5% EtOAc/hexanes as eluent to afford 11(2.72 g, 82%) as light yellow oil. TLC: 10% EtOAc/hexanes, R_(f)≈0.45;¹H NMR (CDCl₃, 400 MHz) δ 5.40-5.34 (m, 2H), 3.64 (s, 3H), 3.26 (t,J=6.7 Hz, 2H), 2.30 (t, J=7.7 Hz, 2H) 2.10-1.98 (m, 4H) 1.66-1.54 (m,2H), 1.48-1.24 (m, 100H).

Methyl 13-(3-pentylureido)tridec-8(Z)-enoate⁶ (12)

Triphenylphosphine (2.7 g., 11.0 mmol) was added to a room temperaturesolution of 11 (1.4 g, 5.24 mmol) in dry THF (25 mL). After 2 h, water(200 μL) was added and the stirring was continued for another 8 h. Thereaction mixture was then diluted with EtOAc (100 mL), washed with water(20 mL) and brine (25 mL). Aqueous layers were back-extracted with EtOAc(2×30 mL). The combined organic extracts were dried over Na₂SO₄,concentrated under reduced pressure and further dried under high vacuumfor 4 h. The crude amine was used in the next step without additionalpurification. Procedure ref.: S. Chandrasekhar; S. S. Sultana; N.Kiranmai; Ch. Narsihmulu Tetrahedron Lett. 2007: 48, 2373.

n-Pentyl isocyanate (0.78 g, 6.9 mmol) was added to a room temperaturesolution of the above crude amine (1.4 g, 5.8 mmol) in dry THF (25 mL).After 6 h, reaction mixture was concentrated under reduced pressure andthe residue was purified by SiO₂ column chromatography using 30%EtOAc/hexanes as eluent to give 12 (1.70 g, 85%) as a colorless, viscousoil. TLC: 50% EtOAc/hexanes, R_(f)≈0.40; ¹H NMR (CDCl₃, 400 MHz) δ5.40-5.26 (m, 2H), 4.46-4.32 (m, NH, 2H), 3.66 (s, 3H), 3.18-3.10 (m,4H), 2.34 (t, J=7.7 Hz 4H), 2.06-1.94 (m, 4H), 1.66-1.56 (m, 2H),1.54-1.42 (m, 14H), 0.88 (t, J=7.0 Hz, 3H).

Methyl 13-(3-pentylureido)tridec-8(Z)-enoic acid⁷ (13)

LiOH (6.2 mL, 2.0 M aqueous solution, 3.0 equiv) was added to a 0° C.solution of 12 (1.80 g, 5.8 mmol) in THF (25 mL) and deionized H₂O (4mL). After stirring at room temperature overnight, the reaction mixturewas cooled to 0° C., the pH was adjusted to 4.0 with 1M aq. oxalic acid,and extracted with ethyl acetate (2×20 mL). The combined extracts werewashed with water (30 mL), brine (25 mL), dried over anhydrous Na₂SO₄,and concentrated in vacuo. The residue was purified by SiO₂ columnchromatography using 25% EtOAc/hexanes as eluent to give 13 (1.48 g,86%) as white solid, m.p.=67.1° C. TLC: 80% EtOAc/hexanes, R_(f)≈0.30;¹H NMR (CDCl₃, 400 MHz) δ 5.40-5.26 (m, 2H), 3.17-3.10 (m, 4H), 2.32 (t,J=6.7 Hz, 2H), 2.09-1.95 (m, 4H), 1.65-1.48 (m, 6H), 1.44-1.22 (m, 12H),0.89 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 178.5, 159.6, 130.5,129.5, 40.9, 40.8, 34.4, 29.9, 29.8, 29.2, 28.7, 28.5, 27.2, 26.7, 24.9,22.6, 14.2.

(S)-2-(13-(3-Pentylureido)tridec-8(Z)-enamido)succinate (14)

L-Aspartic acid dimethyl ester (38 mg, 0.191 mmol) and HATU (67 mg,0.176 mmol) were added to a stirring solution of 13 (50 mg, 0.147 mmol)and DIPEA (74 mg, 0.573 mmol) in anhydrous DMF (2 mL) under an argonatmosphere. After 5 min, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(33.8 mg, 0.176 mmol; EDCI) was added as a solid. After stirring for 12h at room temperature, the reaction mixture was diluted with EtOAc (15mL), washed with water (5 mL), and brine (10 mL). The combined aqueouslayers were back-extracted with EtOAc (3×10 mL). The combined organicextracts were dried over Na₂SO₄, concentrated under reduced pressure,and the residue was purified by SiO₂ column chromatography using 50%EtOAc/hexanes as eluent to give 14 (60 mg, 84%) as viscous oil. TLC: 60%EtOAc/hexanes, R_(f)≈0.35; ¹H NMR (CDCl₃, 400 MHz) δ 6.64 (d, J=7.9 Hz,1H), 5.38-5.30 (m, 2H), 4.90-4.82 (m, 1H), 4.58-4.44 (m, 2H), 3.75 (s,3H), 3.66 (s, 3H), 3.20-3.10 (m, 4H), 3.04 (dd, J_(1=4.3) Hz, J₂=13.1Hz, 1H), 2.84 (dd, J₁=4.6 Hz, J₂=12.8 Hz, 1H), 2.22 (t, J=6.3 Hz, 2H),2.05-1.98 (m, 4H), 1.70-1.60 (m, 2H), 1.50-1.20 (m, 16H), 0.88 (t, J=6.7Hz, 3H).

(S)-2-(13-(3-pentylureido)tridec-8(Z)-enamido)succinic acid (15)

An aqueous solution of LiOH (2 mL, 2 M solution, 6.0 equiv) was added toa 0° C. solution of 14 (60 mg, 0.124 mmol) in THF (8 mL) and deionizedH₂O (2 mL). After stirring at room temperature overnight, the reactionmixture was cooled to 0° C., the pH was adjusted to 4.0 with 1M aq.oxalic acid, and extracted with ethyl acetate (2×10 mL). The combinedextracts were washed with water (5 mL), brine (5 mL), dried overanhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified bySiO₂ column chromatography using 70-90% EtOAc/hexanes as eluent to give15 (48 mg, 85%) as a viscous, colorless oil. TLC: 5% MeOH/EtOAc,R_(f)≈0.20; ¹H NMR (CD₃OD, 400 MHz) δ 5.38-5.30 (m, 2H), 4.72 (t, J=4.3Hz, 1H), 3.12-3.05 (m, 4H), 2.90-2.72 (m, 2H), 2.22 (t, J=7.7 Hz, 2H),2.10-1.98 (m, 4H), 1.60-1.22 (m, 18H), 1.20 (t, J=7.1 Hz, 3H); ¹³C NMR(CDCl₃, 75 MHz) δ 174.9, 173.0, 172.8, 160.1, 129.9, 129.3, 51.8, 49.8,39.8, 39.7, 35.7, 35.6, 29.9, 29.8, 29.5, 29.0, 28.8, 26.9, 26.7, 25.7,22.3, 13.2.

Disodium (S)-2-(13-(3-pentylureido)tridec-8(Z)-enamido)succinate (16)

Sodium bicarbonate (93 mg, 1.1 mmol) was added to a stirring solution of15 (100 mg, 0.22 mmol) in THF/H₂O (4:1, 5 mL) at rt. After 2 h, the THFwas removed in vacuo and the remaining aqueous phase was stirred withSM-2 Bio-Beads (Bio-Rad, 20-50 mesh; 2 g). After 1 h, the Bio-Beads werecollected by filtration on a sintered-glass funnel, washed with water (5mL×2) and finally with 95% ethanol (20 mL×3). Evaporation of the ethanolwashes in vacuo gave 16 (72 mg, 84%) as a white solid, m.p.=258.5° C.TLC: 10% MeOH/CH₂Cl₂, R_(f)˜0.15; ¹H NMR (CD₃OD, 500 MHz) δ 5.25-5.23(m, 2H), 4.40 (t, 1H, J=4.0), 3.01-2.97 (m, 4H), 2.58-2.56 (m, 2H), 2.13(t, 2H, J=7.0), 1.96-1.94 (m, 4H), 1.51-1.47 (m, 2H), 1.30-1.19 (m,16H), 0.83 (t, 3H, J=7.0); ¹³C NMR (75 MHz, CDCl₃) δ 178.52, 178.38,173.99, 160.21, 129.93, 129.37, 52.66, 39.81, 39.74, 36.27, 29.50,28.93, 26.76, 22.34, 13.26.

Example 3 Synthesis ofN-Isopropyl-N-(5-(2-pivalamidobenzo[d]thiazol-4-yloxy)pentyl)heptanamide(MV=EET-B or SRD-2)

As set forth in FIG. 15, the synthesis of EET B is as follows:

5-(tert-Butyldiphenylsilyloxy)pentan-1-ol (2)

Imidazole (0.65 g, 9.60 mmol) was added to a stirring solution ofpentan-1,5-diol (1.00 g, 9.60 mmol) in dry dichloromethane (10 mL) at 0°C. under an argon atmosphere followed by the dropwise addition oftert-butylchlorodiphenylsilane (3.85 mL, 9.60 mmol) in CH₂Cl₂ (2 mL).The reaction was allowed to slowly reach room temperature. After 12hours, the reaction mixture was washed with water (2×30 mL), brine (20mL), dried over Na₂SO₄, and concentrated under reduced pressure. Theresidue which was purified by SiO₂ flash chromatography using 30% ethylacetate/hexane as eluent to furnish 2 (1.35 g, 46%), recovered SM anddi-protected compound. TLC: 30% EtOAc/hexanes, R_(f)˜0.38; ¹H NMR(CDCl₃, 400 MHz) δ 7.67-7.62 (m, 4H), 7.45-7.35 (m, 6H), 3.67 (t, 2H,J=6.0 Hz), 3.52 (t, 2H, J=6.7 Hz), 2.03-1.93 (m, 2H), 1.72-1.64 (m, 4H),1.04 (s, 9H).

tert-Butyl(5-iodopentyloxy)diphenylsilane (3)

Imidazole (310 mg, 4.40 mmol), iodine (440 mg, 3.5 mmol), and a solutionof 2 (1 g, 2.98 mmol) in CH₂Cl₂ (2 mL) were added sequentially to a 0°C. solution of PPh₃ (450 mg, 3.5 mmol) in CH₂Cl₂ (15 mL) and kept in thedark. After 2 h, the reaction mixture was quenched by adding 20% aq.Na₂S₂O₃ (5 mL). The aqueous layer was extracted with CH₂Cl₂ (2×50 mL).The combined organic extracts were washed with brine (30 mL), dried overanhydrous Na₂SO₄, and the solvent was removed in vacuo. The residue waspurified by careful column chromatography using 5% EtOAC/hexane toafford iodide 3 (1.25 mg, 92%). TLC: 10% EtOAc/hexane, R_(f)˜0.85; ¹HNMR (CDCl₃, 400 MHz) δ 7.67-7.62 (m, 4H), 7.45-7.35 (m, 6H), 3.67 (t,2H, J=6.0 Hz), 3.41 (t, 2H, J=6.7 Hz), 2.03-1.93 (m, 2H), 1.72-1.64 (m,4H), 1.04 (s, 9H).

5-(tert-Butyldiphenylsilyloxy)-N-isopropylpentan-1-amine (4)

K₂CO₃ (1.50 g, 11.05 mmol), isopropylamine (0.65 mL, 11.05 mmol) andiodide 3 (2.50 g, 5.83 mmol) in dry THF (15 mL) were heated at 66° C. ina sealed tube under an argon atmosphere. After 12 h, water (5 mL) wasadded to the reaction mixture which was then extracted with EtOAc (3×50mL). The combined organic extracts were dried over MgSO₄ andconcentrated under reduced pressure to afford 4 as a colorless liquid(1.90 g, 92%) sufficiently pure it was used without furtherpurification. TLC: MeOH/CH₂Cl₂ (1:4), R_(f)˜0.30; ¹H NMR (400 MHz) δ7.65-7.67 (m, 4H), 7.30-7.40 (m, 6H), 3.65 (t, J=6.4 Hz, 2H), 2.70-2.82(m, 1H), 2.55 (t, J=7.3 Hz, 2H), 1.50-1.64 (m, 2H), 1.32-1.50 (m, 4H),1.05 (d, J=5.8 Hz, 3H), 1.04 (s, 9H); ¹³C NMR (100 MHz) δ 135.68,134.21, 129.63, 127.70, 63.95, 48.81, 47.64, 32.60, 30.27, 27.0, 23.76,23.15, 19.34.

N-(5-(tert-Butyldiphenylsilyloxy)pentyl)-N-isopropylheptanamide (5)

Heptanoic acid (2.06 g, 15.86 mmol), and diisopropylethylamine (2.72 mL,21.14 mmol; DIPEA) were added to a stirring solution of the amine 4(4.00 g, 10.57 mmol) in anhydrous DMF (20 mL) under an argon atmosphere.After 5 min, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (3.04 g,15.86 mmol; EDCI) was added as a solid. After stirring for 12 h at roomtemperature, the reaction mixture was diluted with EtOAc (100 mL),washed with water (2×30 mL), and brine (20 mL). The combined aqueouslayers were back-extracted with EtOAc (3×30 mL). The combined organicextracts were dried over Na₂SO₄, concentrated under reduced pressure,and the residue was purified by SiO₂ column chromatography using 30%EtOAc/hexanes as eluent to give amide 5 (4.75 g, 91%) as a viscous oil.TLC: EtOAc/hexanes (3:7), R_(f)˜0.60; ¹H NMR (400 MHz, 1:1 mixture ofrotamers) δ 7.65-7.67 (m, 4H), 7.30-7.40 (m, 6H), 4.62-4.72 and3.96-4.80 (m, 1H, rotamers), 3.62 and 3.68 (t, J=4.8 Hz, 2H, rotamers),3.02 and 3.16 (t, J=5.2 Hz, 2H, rotamers), 2.38 and 2.24 (t, J=5.3 Hz,2H, rotamers), 1.50-1.68 (m, 6H), 1.26-1.44 (m, 8H), 1.18 and 1,12, (d,J=7.3 Hz, 6H, rotamers), 1.03 and 1.04 (s, 9H, rotamers), 0.88 (t, J=7.3Hz, 3H); ¹³C NMR (100 MHz, 1:1 mixture of rotamers) δ 173.38, 172.76,135.80, 135.78, 134.36, 134.12, 129.85, 129.73, 127.88, 127.82, 64.22,63.68, 48.43, 45.62, 43.64, 41.27, 34.13, 34.05, 32.61, 32.36, 31.96,31.93, 31.51, 29.63, 29.47, 27.10, 27.03, 25.94, 25.78, 24.03, 23.77,22.81, 21.63, 20.78, 19.47, 14.33, 14.28.

N-(5-hydroxypentyl)-N-isopropylheptanamide (6)

A solution of 5 (4.75 g, 9.02 mmol) and p-toluenesulfonic acid in MeOH(50 mL) was stirred at rt for 12 h, then quenched with solid NaHCO₃ andfiltered. The filtrate was evaporated under vacuum and the residue wasdissolved in ethyl acetate (50 mL). The ethyl acetate layer was washedwith water (2×50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by silicagel flash column chromatography using 50-60% ethyl acetate/hexane aseluent to afford alcohol 6 (2.35 g, 93%) as a colorless, viscous liquid.TLC: EtOAc/hexanes (1:1), R_(f)˜0.30; ¹H NMR (400 MHz, 55/45 mixture ofrotamers) δ 4.58-4.66 and 3.96-4.08 (m, 1H, rotamers), 3.56 and 3.70 (t,J=5.4 Hz, 2H, rotamers), 3.02 and 3.16 (t, J=5.2 Hz, 2H, rotamers), 2.38and 2.26 (t, J=6.3 Hz, 2H, rotamers), 1.50-1.64 (m, 6H), 1.22-1.40 (m,8H), 1.13 and 1.09 (d, J=7.5 Hz, 6H, rotamers), 0.84 (t, J=7.3 Hz, 3H);¹³C NMR (100 MHz, 55/45 mixture of rotamers) δ 172.81, 62.66, 62.60,48.41, 45.55, 43.56, 41.04, 34.08, 34.0, 32.46, 32.43, 31.86, 31.57,29.40, 25.87, 25.70, 23.77, 23.73, 22.75, 21.57, 20.72, 14.26.

N-(5-Bromopentyl)-N-isopropylheptanamide (7)

TPP (4.45 g, 9.34 mmol) was added to a 0° C. solution of alcohol 6 (2.00g, 7.78 mmol) in dry CH₂Cl₂ (50 mL). After 10 min, CBr₄ (3.10 g, 9.34mmol) was added and the stirring was continued at 0° C. After 2 h, water(20 mL) was added and the reaction mixture was extracted with EtOAc(3×50 mL). The combined extracts were washed with water (2×20 mL), brine(20 mL), dried over Na₂SO₄, and concentrated under reduced pressure. Theresidue was purified by SiO₂ column chromatography using 30-35% ethylacetate/hexane to afford 7 (2.20 g, 91%). TLC: EtOAc/hexanes (3:7),R_(f)˜0.40; ¹H NMR (400 MHz, 45/55 mixture of rotamers) δ 4.60-4.70 and3.96-4.10 (m, 1H, rotamers), 3.46 and 3.36 (t, J=5.8 Hz, rotamers),3.02-3.10 (m, 2H), 2.30 and 2.22 (t, J=7.9 Hz, 2H), 1.80-1.97 (m, 2H),1.40-1.70 (m, 6H), 1.20-1.40 (m, 6H), 1.16 and 1.10, (d, J=7.3 Hz, 6H,rotamers), 0.86 (t, J=7.3 Hz, 3H); ¹³C NMR (100 MHz, rotamers) δ 172.79,172.39, 48.17, 45.47, 43.32, 40.70, 33.89, 33.87, 33.49, 32.48, 32.30,31.81, 31.78, 30.78, 29.27, 28.74, 26.05, 25.84, 25.69, 25.54, 22.64,21.47, 20.62, 14.17.

Isopropyl-N-(5-(2-pivalamidobenzo[d]thiazol-4-yloxy)pentyl)heptanamide(11)

A solution of bromide 7 (0.35 g, 1.09 mmol),N-(4-hydroxybenzo[d]thiazol-2-yl)pivalamide (10) (0.27 g, 1.09 mmol),and K₂CO₃ (0.30 g, 2.18 mmol) in DMF (10 mL) was heated at 80° C. After4 h, the reaction mixture was cooled to rt, water (5 mL) was added andthe mixture was extracted using ethyl acetate (3×30 mL). The combinedorganic extracts were washed with H₂O (2×20 mL), brine (20 mL), driedover Na₂SO₄, and filtered. The filtrate was evaporated under reducedpressure and the residue was purified by silica gel columnchromatography (50-70% EtOAc/hexanes) to give 11 (0.39 g, 72%) as aviscous liquid. TLC: EtOAc/hexanes (7:3), R_(f)˜0.26; ¹H NMR (400 MHz,45/55 mixture of rotamers) δ 7.24 and 7.20 (d, J=7.4 Hz, 1H, rotamers),7.16 and 7.14 (dd, J=7.4 Hz, J=7.4 Hz, 1H, rotamers), 6.90 and 6.80 (d,J=7.4 Hz, 1H, rotamers), 4.58-4.78 and 3.96-4.10 (m, 1H, rotamers), 4.02(t, J=6.3 Hz, 2H), 3.12 (t, J=7.3 Hz, 2H), 2.30 and 2.27 (t, J=5.6 Hz,2H, rotamers), 1.82-1.92 (m, 2H), 1.44-1.70 (m, 4H), 1.38-1.32 (m, 8H),1.20 (d, J=6.4 Hz, 3H), 1.13 (d, J=6.4 Hz, 3H), 1.11 (d, J=6.4 Hz, 3H),0.88-085 (m, 6H); ¹³C NMR (100 MHz, 45/55 mixture of rotamers) δ 189.64,189.62, 173.04, 172.57, 172.55, 167.16, 167.09, 167.06, 146.85, 146.77,146.67, 128.90, 128.87, 125.90, 125.79, 124.24, 124.13, 115.32, 115.06,111.00, 110.09, 110.05, 68.97, 68.74, 58.50, 48.34, 45.62, 41.24, 41.10,40.95, 34.10, 29.52, 29.15, 25.72, 24.28, 21.66, 20.76, 14.30.

N-(4-hydroxybenzo[d]thiazol-2-yl)pivalamide (10)

To a suspension of 2-aminobenzo[d]thiazol-4-ol (0.50 g, 3.01 mmol) intoluene (10 mL) was added trimethyacetyl chloride (3.60 mL, 30.10 mmol)at room temperature. The reaction mixture was stirred at 115° C. for 22h. The solvents were evaporated and the residue was azeotroped withEtOAc to give 9 (0.78 g, 78%) as a tan solid. The suspension of theabove solid (0.78 g) in MeOH (15 mL) was treated with K₂CO₃ (0.20 g,3.50 mmol) and stirred at room temperature for 6 h. MeOH was evaporatedand the residue was diluted with H₂O. The resulting mixture wasneutralized with concd HCl to pH=7 and extracted with EtOAc (3×30 mL).The combined organic phases were dried over sodium sulfate, filtered,and concentrated in vacuo to provide 10 (0.48 g, 82%) s a tan solid.TLC: EtOAc/hexanes (7:3), R_(f)˜0.32; ¹H NMR (400 MHz, CDCl₃) δ 10.25(br s, 1H), 9.66 (br s, 1H), 7.33 (d, J=7.4 Hz, 1H), 7.09 (dd, J=7.4,7.4, Hz, 1H), 6.82 (d, J=7.4 Hz, 1H), 1.45 (s, 9H). ¹³C NMR (100 MHz,CDCl₃) δ 177.15, 159.12, 148.10, 136.55, 133.00, 125.55, 113.40, 111.50,39.70, 27.38.

Example 4 In Vitro Screening of EET Analog Library

In this Example, the inventors performed both vascular relaxation assaysand a soluble epoxide hydrolase inhibition assay using the newlysynthesized compounds. The results of these assays are recorded in thelast three columns of Table 1 above and FIG. 14. As a result of theseassays, four compounds, compounds 26, 20, 7 and 30 were selected forfurther in vivo testing, as outlined in later Examples.

Soluble Epoxide Hydrolase Inhibition.

Compounds were tested for their ability to inhibit recombinant solubleepoxide hydrolase (sEH) protein. The assay utilizes(3-Phhenyl-oxiranyl)-acetic acidcyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), a sensitivesubstrate for sEH that can be used to monitor the activity of both humanand murine enzymes. Hydrolysis of the substrate epoxide yields a highlyfluorescent product, 6-methoxy-2-Naphthaldehyde, which can be monitoredat excitation and emission wavelengths of 330 and 465 nm, respectively.See Wolf et al., Anal Biochem 355:71-80, 2006 PMID: 16729954. Humanrecombinant sEH was incubated with substrate and compounds ranging inconcentration from 0.1 to 1000 nM. The percent activity remaining ateach concentration was plotted and an IC50 (concentration at which thereis 50% inhibition) determined utilizing statistical software.

Vasodilator Activity.

Vasorelaxant activity was measured in bovine coronary artery. Bovinehearts were obtained and the left anterior descending coronary arterywas dissected and cleaned of connective tissue. Vessels of 1 mm diameterwere cut into rings of 3 mm width as previously described (3, 27, 39).Vessels were stored in Krebs buffer consisting of (in mM) 119 NaCl, 4.8KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, 11 glucose, 0.02 EDTA, and 3.2CaCl₂. The vessels were suspended from a pair of stainless steel hooksin a 6-ml water-jacketed organ chamber. The organ chamber was filledwith Krebs buffer and bubbled with 95% O₂-5% CO₂ at 37° C. One hook wasanchored to a steel rod and the other hook to a force transducer (modelFT-03C; Grass Instruments, West Warwick, R.I.). Tension of the vesselwas measured by an ETH-400 bridge amplifier, and the data were acquiredwith a MacLab 8e analog-to-digital converter and MacLab software version3.5.6 (AD Instruments, Milford, Mass.) and stored on a Macintoshcomputer for subsequent data analysis.

Basal tension was set at the length-tension maximum of 3.5 g andequilibrated for 1.5 h. KCl (40 mM) was added to the chamber untilreproducible maximal contractions were maintained. U-46619 (10-20 nM), athromboxane receptor agonist, was used to precontract the vessels frombasal tension to between 50% and 90% of the maximal KCl contraction.Cumulative additions of compounds were added to the chamber. Betweenconcentration-response curves, the chambers were rinsed with fresh Krebsbuffer, 40 mM KCl was administered to determine the maximum contraction,and the vessels were rinsed. Consecutive concentration-response curveswere performed with 14,15-EET followed by a concentration-response curveto a compound. The experiment was always repeated with the order of theagonists reversed. In control experiments with consecutiveconcentration-response curves to 14,15-EET, the secondconcentration-response curve with compound was identical to the first.Tension was represented as percent relaxation where 100% relaxation wasbasal pre-U-46619 tension. The relaxation was plotted versus compoundconcentration and the EC50 determined utilizing statistical software.

Results of Vasodilator and sEH Inhibitory Activity Assays.

The results of vasorelaxant and sEH inhibitory activities of the 33synthesized compounds are summarized in Table 1. Using thepharmacophoric moiety of EET, a number of EET analogs were designed withimproved solubility and resistance to auto-oxidation, etherification andmetabolism by soluble epoxide hydrolase (sEH). It is observed that thesecompounds possess activity analogous to EET as evident from theirvasorelaxant activity in bovine coronary artery and sEH inhibitory(sEHi) activity. Among these, four compounds among those that weredesigned by replacing COOH group of the EET pharmacophore with isostericreplacement or a heterocyclic surrogate were studied for potentialantihypertensive effect. The results of vasorelaxant and sEH inhibitoryactivities of these compounds are summarized in Table 2 below.

TABLE 2 Characteristics of the compounds selected for testing in the invivo models. Vascular relaxation SEHi % activity relaxation EC₅₀ IC₅₀Compound Structure (10 μM) (μM) (nM) SRD-I-71-9

109 .32 >500 LGK-I-119-15

119 0.18 11 JLJ-I-94-6

91 1.6 392 MV-IV-110-20

96 1.3 >500

Example 5 In Vitro Testing of Four Compounds Using Rat Models ofHypertension

Telemetry Blood Pressure Measurement. To accurately detect changes inblood pressure and heart rate, telemetry transmitters (Data SciencesInc., St. Paul, Minn.) were implanted in rats one week prior to theexperimental period according to manufacturer's specifications whileunder pentobarbital anesthesia. In brief, an incision was made to exposethe femoral artery that was occluded to allow insertion of thetransmitter catheter. The catheter was secured in place with tissue glueand the transmitter body was sutured in place and the incision line wasclosed. Rats were allowed to recover from surgery and were returned toindividual housing. A baseline arterial pressure was recorded for priorto the experimental period. Mean arterial pressure was continuouslyrecorded throughout the experimental period.

Angiotensin Hypertension.

Telemetry transmitters were implanted into male Sprague-Dawley rats(225-275 g) as described. After recording basal blood pressure, osmoticpumps were implanted (s.c.) to deliver angiotensin at a dose of 60ng/min. EET analogs were administered by an osmotic pump (2 mg/d, i.p.)and blood pressure was continuously monitored.

Spontaneously Hypertensive Rats (SHR).

Telemetry transmitters were implanted into male SHR as described. Afterthe surgical recovery period, baseline mean arterial pressure wasrecorded. In this series of experiments, EET analogs were administeredby osmotic pump (2 mg/d, i.p.) and blood pressure was continuouslymonitored.

Protein Excretion Measurements.

Animals were placed in a metabolic cage and urine was collected in aconical tube. Samples were stored at −80° C. until assayed. Urinaryprotein excretion was assessed as an index for renal injury. Protein wasdetermined by the Bradford colorimetric method and creatinine wasdetermined by the picric acid colorimetric method.

Telemetry and urinary analysis methods are further outlined in thefollowing publications: Imig J D, Zhao X, Zaharis C Z, Olearczyk J J,Pollock D M, Newman J W, Kim I H, Hammock B D. An orally active epoxidehydrolase inhibitor lowers blood pressure and provides renal protectionin salt-sensitive hypertension. Hypertension 46:975-981, 2005. PMID:1615779; Elmarakby A A, Quigley J E, Olearczyk J J, Srindhar A, Cook AK, Inscho E W, Pollock D M, Imig J D. Chemokine receptor 2b blockadeinhibition provides renal protection in angiotensin II-salthypertension. Hypertension 50:1069-1076, 2007. PMID: 17938380; andOlearczyk J J, Quigley J E, Mitchell B, Yamamoto T, Kim I H, Newman J W,Lauria A, Hammock B D, Imig J D. Inhibition of the soluble epoxidehydrolase protects the kidney from damage in hypertensive Goto-Kakizakirats. Clinical Science 116:61-70, 2009. PMID: 18459944. Thesepublications are incorporated by reference herein.

Statistical Analysis.

All data are presented as mean±SEM. Mean arterial blood pressure datawere analyzed using analysis of variance (ANOVA) for repeatedmeasurements. Differences were considered statistically significant withp<0.05 compared to the control. Analyses were performed using GraphPadPrism Version 4.0 software (GraphPad Software Inc, La Jolla, Calif.).

Results-Effects on Blood Pressure and Heart Rate.

Spontaneously hypertensive rat (SHR). In this model of hypertension,blood pressure lowering abilities of four selected compounds werestudied. It is observed that two of these four compounds had bloodpressure lowering effects. SRD (chemical structure shown in FIG. 1A) andLGK (chemical structure shown in FIG. 2A) lacked blood pressure loweringactions in SHR. In the SRD treated SHR group, after two weeks oftreatment the blood pressure was similar to the vehicle treated SHRgroup (150±5.0 vs. 141±3.0 mmHg) (see FIG. 1B-C). After two weeks oftreatment, LGK did not change the blood pressure (137±1.0 vs. 141±3.0mmHg) compared to the vehicle in SHR (FIG. 2 C-D). Similar to theireffects on blood pressure, neither SRD nor LGK affected the heart rate(SRD, 344±23.0 vs. 331±17.0 BPM; LGK, 325111.0 vs. 331±17.0 BPM)compared to vehicle SHR.

Two weeks treatment with JLJ (chemical structure shown in FIG. 3A)caused a moderate decrease in blood pressure in SHR compared to vehicletreated group (131±2.0 vs. 141±3.0 mmHg) and its blood pressure loweringeffect has been seen from the first week of the treatment (FIG. 3B-C).The compound MV (chemical structure shown in FIG. 4A) also demonstratesa similar blood pressure lowering effect in SHR and caused a 12 mmHgdecrease in blood pressure compared to vehicle (129±2.0 vs. 141±3.0mmHg). Moreover, similar to JLJ, it was observed that MV started tolower blood pressure within four days of the treatment in SHR andmaintained this effect until the end of the two-week treatment period(see FIG. 4B-C). In contrast to their blood pressure lowering effect itwas further observed that, neither JLJ nor MV had any affect on theheart rate (JLJ, 316±23.0 vs. 331±17.0 BPM; MV, 318±24.0 vs. 331±17.0BPM) compared to vehicle SHR. Considering promising blood pressurelowering effects of JLJ and MV in SHR model, we have further testedthese compounds in another model of hypertension, angiotensin II (AngII) hypertension.

Ang II Hypertensive Rats.

The compound JLJ demonstrates an attenuating effect on the Ang IIinduced elevation in blood pressure from the beginning of the treatmentand this was maintained throughout the treatment period. It is observedthat at the end of two-week of treatment period JLJ markedly attenuatedthe Ang II induced hypertension compared to vehicle (135±5.0 vs. 150±3.2mmHg) (FIG. 5A-B). Similar to JLJ, the compound MV also demonstratesmarked attenuating effect on the Ang II hypertension (107±2.0 vs.150±3.2 mmHg) and this attenuating effect was observed throughout thetreatment period (FIG. 6A-B). Similar to the SHR, in ANG II hypertensionneither JLJ (410±25.0 vs. 396±25.0 BPM) nor MV (385±16.0 vs. 396±25.0BPM) demonstrates any effect on the heart rate compared to vehicle aftertwo weeks of treatment.

Effects of MV on Sodium Excretion and Protein Excretion in Ang IIHypertension.

In the present study we have observed that the two weeks treatment withcompound MV (chemical structure shown in FIG. 4A) caused natriuresiscompared to vehicle (2.7±0.3 vs. 1.9±0.7 mmol/d) in Ang II hypertension.It is also observed that the compound MV decreased the urinary proteinto creatinine ratio (1.5±0.2 vs. 2.8±0.7), an indicator of renal injury,in Ang II hypertension.

Example 6 Effect of EET Analogs in Treating Cisplatin Nephrotoxicity

In this Example, the inventors investigated the kidney protective effectof two newly developed orally active EET analogs in cisplatin-inducednephrotoxicity. It was demonstrated that EET analogs offered markedreno-protection during cisplatin administration and this effect wasrelated to their anti-oxidative, anti-inflammatory, anti-ER stress andanti-apoptotic activities. We have further demonstrated that whileprotecting the kidney from the deleterious nephrotoxic effect ofcisplatin, these EET analogs did not compromise cisplatin'schemotherapeutic effect.

Nephrotoxicity severely limits the use of the anti-cancer drugcisplatin. Oxidative stress, inflammation and endoplasmic reticulum (ER)stress contribute to cisplatin-induced nephrotoxicity. We developedorally active EET analogs (including without limitation compounds EET-A& EET-B) by modifying the carboxylate, olefins, and epoxide moieties ofEET pharmacophore. We determined if administering the claimed EETanalogs would decrease nephrotoxicity, including cisplatin-inducednephrotoxicity. Cisplatin was administered (7 mg/kg i.p.) in ratspretreated for 7 days with EET analogs (10 mg/kg/d p.o., n=5) or vehicle(n=7). On day 5 following cisplatin injection, urine, plasma, andkidneys were collected. Cisplatin-induced nephrotoxicity was manifest bya 3-5-fold increase in BUN, plasma creatinine (PCr), urinaryN-acetyl-(D)-glucosaminidase activity (NAG), kidney injury molecule-1(KIM-1), and renal tubular cast formation. EET analogs attenuatedcisplatin-induced increases in BUN (vehicle: 241±51 vs. EET-A: 108±30 &EET-B: 120±33 mg/dL), PCr (3.1±0.2 vs. 2.0±0.2 & 1.4±0.2 mg/dL), KIM-1(296±94 vs. 85±29 & 57±13 ng/d), and NAG (3.0±0.6 vs. 0.5±0.1 & 0.6±0.2U/d) (P<0.05). Cisplatin-induced renal tubular cast formation wasreduced 50% by EET analog treatment. EET analogs attenuatedcisplatin-induced kidney TBARS formation (vehicle: 16±2 vs. EET-A: 7±1;EET-B: 8-1 μmol/g) and cause 2-3-folds decrease in kidney expression ofNOX1 and gp91phox mRNAs (P<0.05). Cisplatin-induced nephrotoxicity wasaccompanied by elevated renal inflammation and ER stress resulting inincreased kidney mRNA expression of inflammatory (TNF-α, IL-6, IL-1β)and ER stress (caspase 12, GRP78) genes. EET analogs caused 30-70%reductions in the expression of these inflammatory and ER stress genes(P<0.05). Cisplatin caused apoptotic signalling in the kidney withelevated Bak/Bcl2 and Bax/Bcl2 mRNA expression ratios and renal corticalcaspase 3 activity. EET analogs caused 2-14-folds reduction in kidneyBak/Bcl2 and Bax/Bcl2 mRNA expression ratios as well as a 50% reductionin renal caspase 3 activity (P<0.05). In an in vitro study with severalcancer cell-lines, we also demonstrate that EET analog's kidneyprotective effects dose not compromise cisplatin's anti-cancer property.Collectively, these data demonstrate that orally active EET analogsprotect from nephrotoxcity, including cisplatin-induced nephrotoxicity,by reducing oxidative stress, inflammation, and ER stress withoutaffecting cisplatin's chemotherapeutic effects. In addition, the EETanalogs will also protect against other common cisplatin side effects,including loss of hearing.

In Vivo Animal Experiments.

Experiments were approved and carried out according to the guidelines ofthe Institutional Animal Care and Use Committee, Medical College ofWisconsin, Milwaukee, USA. Male Wistar-Kyoto (WKY) rats weighing 180-200g (Charles River, Mass., USA). All animals were kept in atemperature-controlled environment with a 12-h light/dark cycle and wereallowed free access to food and water at all times. An acclimatizationperiod of 6 days was allowed for the rats before experimentation. Therats were assigned into four groups. Group 1 (WKY, n=5-7): Rats receiveddrinking water ad libitum for seven days and on day 7 DMSO (SigmaAldrich, St. Louis, Mo., USA) was administered (300-500 μl i.p). DMSOwas used to prepare the cisplatin (CP) (Sigma Aldrich, St. Louis, Mo.,USA) solution used in this study, and the maximum volume of theinjection set at 500 μl. Group 2 (CP+Vehicle, n=5-7): Rats werepretreated with vehicle (0.05% ethanol and 0.1% PEG-400 v/v) in drinkingwater for seven days and then on day 7 CP was administered (7 mg/kgi.p.) followed by another five days treatment with vehicle. Group 3(CP+EET-A, n=5-7): These rats are pretreated with the EET analog EET-A(10 mg/kg/day p.o.) for seven days in drinking water and then on day 7administered CP as a single injection (7 mg/kg i.p.) followed by anotherfive days treatment with EET-A. Group 4 (CP+EET-B, n=5-7): Rats of thisgroup are pretreated with another EET analog EET-B (10 mg/kg/day) for 7days in drinking water and then on day 7 CP was administered as a singleinjection (7 mg/kg i.p.) followed by another five days treatment withEET-B. Rats of groups 2, 3 and 4 had free access to vehicle, EET-A and-B in drinking water, respectively. One day before the rats weresacrificed, urine of each rat was collected over a 24-h period, and thevolume was measured. Five days after CP or DMSO administration, ratswere anesthetized for blood sample collection followed by euthanasia andtissue collection. Urine and plasma samples were kept frozen at −80° C.until analyzed. The kidneys were removed, washed with physiologicalsaline and stored at −80° C. until used for RT-PCR analysis,thiobarbituric acid reactive substance (TBARS) measurement and caspase 3activity assay. A part of the kidney also preserved in 10% bufferedformalin for histological examination.

Biochemical Analysis.

The levels of blood urea nitrogen (BUN) (BioAssay Systems, Hayward,Calif., USA) and serum creatinine (Cayman Chemical Company, Ann Arbor,Mich., USA) were measured spectrophotometrically using commercial kits.Urinary content of creatinine and protein were measured using commercialkits (Cayman Chemical Company, Ann Arbor, Mich., USA), and the activityof urinary N-acetyl-b-glucosaminidase (NAG) in the urine was measured bya kit from Diazyme (Diazyme Laboratories, Poway, Calif., USA). Whileurine content of kidney injury molecule-1 (KIM-1) was measured usingELISA (R&D Systems, Inc. Minneapolis, Minn., USA).

Determination of Malondialdehyde in the Kidney.

Malondialdehyde (MDA) is a thiobarbituric acid reactive substance(TBARS) that is formed as an end-product of lipid peroxidation andserves as an important index of oxidative stress. To determine thekidney MDA level, the rat kidney was homogenized with buffer containing1.5% potassium chloride to obtain a 1:10 (w/v) whole kidney homogenate.Using a commercially available kit (Cayman Chemical Company, Ann Arbor,Mich., USA), MDA was measured spectrophotometrically after reaction withthiobarbituric acid.

Determination of Caspase 3 Activity.

Caspase 3 activity in the kidney homogenate was determined using acommercial fluorimetric assay kit (Sigma Aldrich, St. Louis, Mo., USA).Kidney homogenate was prepared with a lysis Buffer (50 mM HEPES, pH 7.4,with 5 mM CHAPS and 5 mM DTT). Kidney homogenate was centrifuged at10,000 g for 10 min and the resulting supernatant was used for theassay. The caspase 3 fluorimetric assay is based on the hydrolysis ofthe peptide substrate acetyl-Asp-Glu-ValAsp-7-amido-4-methylcoumarin(Ac-DEVD-AMC) by caspase 3, resulting in the release of the fluorescent7-amino-4-methylcoumarin (AMC) moiety. The caspase 3 activity isexpressed as nmol of AMC/min/L.

Real-Time PCR Analyses.

Real-Time analysis was carried out to assess the expression of oxidative(gp91phox, NOX1, SOD1, SOD2, SOD3), inflammatory (TNF-α, IL-6, IL-1β),apoptotic (Bax, Bak, Bcl-2) and endoplasmic reticulum stress (GRP78,caspase 12) related genes in the kidney. Total RNA was isolated fromkidney homogenate using TRIzol LS reagents (Invitrogen LifeTechnologies, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The isolated RNA was treated with RNase-free DNase(Invitrogen, Carlsbad, Calif., USA) to remove traces of genomic DNAcontamination. The mRNA samples were quantified by spectrophotometry at260 nm and 1 μg of total RNA was reverse-transcribed to cDNA usingiScript™ Select cDNA Synthesis Kit (Bio-Rad, Hercules, Calif., USA).

The target gene expression was quantified by iScript One-Step RT-PCR Kitwith SYBR green using MyiQ™ Single Color Real-Time PCR Detection System(Bio-Rad Laboratories, Hercules, Calif., USA). Each amplified sample inall wells was analyzed for homogeneity using dissociation curve analysisusing iQ5 Optical System Software, Version 2.1 (Bio-Rad Laboratories,Hercules, Calif., USA). After denaturation at 95° C. for 2 min, 40cycles were performed at 95° C. for 10 s and at 60° C. for 30 s. Eachsample was run in triplicate, and the comparative threshold cycle(C_(t)) method was used to quantify fold increase (2^(−ΔΔCt)) in theexpression of the target genes compared to controls. In analyzing therelative expression of the target genes, the C_(t) values werenormalized to a housekeeping gene (pgk1). Statistical analyses werecarried out for at least 5-7 experimental samples in each experimentalgroup. Primers used in this study were designed based on several earlierreports. After fixation of the kidneys with 10% buffered formalin, renaltissues were sectioned and stained with periodic acid-Schiff (PAS)reagents for histological examination. The numbers of tubules thatcontain proteinaceous casts were determined at magnification of ×200 toassess tubular damage using an image analyzing software NIS Elements ARversion 3.0 (Nikon instruments inc., Melville, N.Y., USA). Thepercentage area positive for cast was calculated from the mean of eightcortical and five medullary fields (×200) for each kidney sample. Tominimize observer bias, the cast area calculation was performed in ablinded fashion without knowledge of the treatment group from which thetissues are originated.

In Vitro Anti-Tumor Activity of Cisplatin in the Presence and Absence ofEET Analog.

In this study, HEK293, U87MG, Hela cell-lines were obtained from theATCC (Manassas, Va., USA) (HEK293, U87MG, Hela), and NCCIT was collectedfrom Department of Pediatrics, UT Southwestern Medical Center at Dallas,Tex., USA. All cell lines were maintained in DMEM or RPIM with 10% fetalbovine serum and penicillin/streptomycin purchased from LifeTechnologies (Grand Island, N.Y., USA). Cisplatin was purchased fromeither Sigma (St. Louis, Mo., USA) or CalBiochem/EMD Biosciences(Billerica, Mass., USA). Cells were seeded in 96-well plates at 500 to4,000 cells per well depends on cell type. Twenty-four hours later, thecells were treated with cisplatin or vehicle and/or the EET analogueEET-A at various concentrations for 72 h. Cell viability was measured byalamar blue assay using resazurin (Sigma Aldrich) according to themanufacturer's guidelines. Viability results were measured byfluorescence/absorbance in a 96-well plate reader from BMG Labtech(Cary, N.C., USA) and the IC₅₀ was calculated by GraphPad Prism®software (GraphPad Software Inc, La Jolla, Calif., USA).

Statistical Analysis.

Results are reported as mean±S.E.M. Statistical significance between twomeasurements was determined by the two-tailed unpaired Student's t test(and among groups it was determined by repeated measure one-way analysisof variance followed by Tukey's post-hoc test) by using GraphPad Prism®Version 4.0 software (GraphPad Software Inc, La Jolla, Calif., USA).Probability values of P<0.05 were considered significant where thecritical value of P was two-sided.

Results. EET Analog Treatment Attenuates Renal Dysfunction and Injury inCisplatin Administered Rats.

To investigate the effects of EET analogs in cisplatin (CP)-inducedrenal dysfunction, levels of urea (blood urea nitrogen or BUN) andcreatinine were measured in the serum of both EET analog-treated and-untreated rats after five days of the CP administration. As shown inthe FIG. 8, CP administration caused 3 and 9-fold increase in the serumcreatinine and BUN levels (FIGS. 7 a and 7 b), respectively (P<0.05).Treatment with EET analogs (EET-A and -B) resulted in 30-50% reductionsin the elevated levels of serum creatinine and BUN in rats administeredwith CP compared to those given vehicle (DMSO) (P<0.05). To determinethe effects of EET analogs in CP-induced renal dysfunction, we furtherstudied urinary excretion of KIM-1, NAG and protein after five days ofCP administration (FIGS. 7 c and 7 d). There were 5 and 10-foldincreases in urinary excretion of NAG and KIM-1 in the CP-administeredrats compared to vehicle-administered controls (P<0.05). Moreover, wealso demonstrated that cisplatin-administration caused markedproteinuria compare to vehicle administration (vehicle vs. cisplatin,25.7±1 vs. 53±5.1 mg/d, P<0.05). Both EET analogs, EET-A and EET-Bresulted 30-50% reduction in the urinary excretion of NAG and KIM-1compared to CP-administered rats treated with vehicle (P<0.05) (FIGS. 7c-d).

We have also observed at least a 40% reduction of cisplatin-inducedproteinuria by both EET analogs (Vehicle vs. EET-A and -B; 53±5 vs. 33±8and 32±3 mg/d, P<0.05). In the present study the CP-induced kidneydysfunction was further assessed using histological examination of thekidney. Administration of CP resulted in tubular injury as manifested bya vacuolation and desquamation of the renal epithelial cells along withsevere intra-tubular proteinaceous cast formation in both the corticaland medullary regions of the kidney compared to vehicle-administeredrats. Both EET analogs protected the kidney in CP-administered rats with≧50% reduction of the tubular cast area in cortex and medulla comparedto CP-administered rats treated with vehicle (P<0.05) (FIGS. 8 a-b).

EET Analog Treatment Attenuates Cisplatin-Induced Renal OxidativeStress, Inflammatory Response and Endoplasmic Reticulum Stress.

Real-Time PCR analysis of the mRNA expressions of NADPH oxidase subunitsNOX1 and gp91phox (FIG. 9) demonstrated increased expression of theseoxidative marker genes in cisplatin (CP) administered rats (P<0.05).There was 2-3-folds attenuation in the cisplatin-induced increase in therenal expression of NOX1 and gp91 phox mRNA were reduced by EET analogsA and B (P<0.05) (FIG. 9 a-b). CP-administration also resulted in amarked elevation in the kidney content of melondialdehyde (MDA), whichis one of the important indicators of oxidative stress. Treatment withEET analogs caused 50% reduction of MDA level in the kidney ofCP-administered rats (P<0.05) (FIG. 9 c). It was further observed thatadministration of CP resulted in 2-5-folds reductions in the mRNAexpression of superoxide dismutase (SOD) 1 and SOD3 (P<0.05) whileexpression of SOD2 was unchanged. Treatment with EET analogs caused2-3-folds increase in the expression of SOD1 in the CP-administered rats(P<0.05) while the expression of SOD3 remained unaltered across theexperimental groups (FIGS. 9 d-f).

To investigate the effect of EET analogs on the CP-induced inflammationthat is associated with renal dysfunction, we studied the renalexpression of mRNAs that code for tumour necrosis factor-α (TNF-α),interleukin-6 (IL-6) and interleukin-1β. These variables demonstrated a2-50-fold increase in their expression in the vehicle treatedCP-administered rats compared to the rats administered vehicle (P<0.05)(FIGS. 10 a-c). Treatment with both EET analogs (EET-A and B) resultedin 40-60% reductions in the renal mRNA expressions of all theinflammatory markers in CP-administered rats (all P<0.05).

We have also observed 4-fold increase in the mRNA expressions of ERstress markers GRP78/BiP and caspase 12 in the vehicle treatedCP-administered rats compared to vehicle-administered rats (P<0.05)(FIGS. 11 a-b). In CP-administered rats, treatment with both EET analogs(EET-A and B) caused 2-4-fold reduction in the elevated renalexpressions of GRP78 and caspase 12 mRNAs compared to vehicle treatment(P<0.05) (FIGS. 11 a-b).

EET Analog Treatment Attenuates Cisplatin-Induced Renal Apoptosis.

There was a 70% reduction in the renal expression of Bcl-2 mRNA in thevehicle treated CP-administered rats compared to rats administeredvehicle (P<0.05) (FIG. 12 a). EET analog treatment caused 2-10-foldincrease in the expression of the anti-apoptotic Bcl-2 in theCP-administered rats compared to vehicle treated CP-administered rats(P<0.05) (FIG. 12 a). Moreover, CP administration resulted in 4-20-foldraise in the Bax/Bcl-2 and Bak/Bcl-2 ratios, and therefore indicatedelevated apoptotic signalling in the CP-administered rats (FIGS. 12 b-c)(P<0.05). EET analogs treatment caused 2-3-fold reduction in Bax/Bcl-2and Bak/Bcl-2 ratios compared to CP-administered rats treated withvehicle (P<0.05) (FIGS. 12 c-d). The CP-induced elevated apoptoticsignalling was further characterized with higher caspase 3 activity(FIG. 12 d) in CP-administered rats compared to the rats administeredvehicle (P<0.05). Treatment with EET analogs attenuated such CP-inducedcaspase 3 activity by 50% compared to the CP-administered rats treatedwith vehicle (P<0.05) (FIG. 12 d). These results clearly demonstratedattenuation of CP-induced apoptotic signaling in the presence of EETanalog treatment.

EET Analog Treatment Dose not Compromise the Chemotherapeutic Effect ofCisplatin.

We demonstrate that in three different cancer cell lines, Hela, NCCITand U87 cisplatin markedly inhibit the cell growth with IC₅₀ ranged from1.1-9.24 μM (FIG. 13 a). In a similar approach with these cell lines,EET-A had no observable effects on cell number (FIG. 13 b). Moreover,concurrent application of EET-A and cisplatin did not influence thecisplatin's chemotherapeutic effect neither on the normal kidney cells(data not shown) nor on the NCCIT cancer cell line (FIG. 13 c). It isdemonstrated that when cisplatin and EET-A were used concurrently, theIC₅₀ for cisplatin was 2.60, 2.55, and 2.44 μM with 0, 1, and 10 ng/mlEET in NCCIT cells.

Discussion.

A critical limitation of cisplatin chemotherapy is the induction oftubulointestinal inflammation, renal oxidative stress, ER stress andtubular cell apoptosis that lead to acute kidney injury. It is reportedthat 40% cancer patients who treated with cisplatin develops acute renalinjury. Unfortunately, efficient pharmacotherapies to attenuate thisdebilitating complication of a widely used chemotherapy like cisplatinare not available. In an attempt to contribute to this area, currentstudy investigated the kidney protective effect of chronic treatment ofepoxyeicosatrienoic acid (EET) analogs on cisplatin-inducednephrotoxicity.

There is strong evidence that EET analogs have ability to protect organby mechanisms involving its anti-inflammatory, anti-apoptotic andanti-oxidative activities. With this background, in the present study wehypothesized that with its strong organ protective ability, EET willprotect the kidney from cisplatin nephrotoxicity. In our attempt, wehave synthesized two novel EET analogs and investigated their kidneyprotective effects in cisplatin-induced nephrotoxicity using aclinically relevant approach with chronic administration of EET analogsin drinking water to the rat. We demonstrate that a singleadministration of cisplatin caused marked renal injury evident fromincreased PCr, BUN, urinary excretion of renal tubular injury markerslike NAG and KIM-1 along with marked proteinuria and tubular castformation. Our results supports several earlier studies reportedcisplatin-induced nephrotoxicity in pre-clinical animal models.Interestingly, we also demonstrate that the chronic treatment with EETanalogs in drinking water markedly protected the kidney fromcisplatin-induced nephrotoxic injury with reductions in all renal injurymarkers studied in this study. In relation to our approach in thepresent study, a recent study demonstrated that acute administration ofsEH inhibitor could reduce cisplatin-induced renal dysfunction in mice.However, it is known that current sEH inhibitors are limited in effectsas they undergo metabolism and incorporation into the membrane,³⁶ thusindicates a limitation of this finding in clinical translationalimplication. Moreover, the study carried out by Parrish et al.³⁵ did notprovide evidence on the possible mechanism by which EET or sEH inhibitorreduces renal dysfunction in cisplatin-induced nephrotoxicity.

Currently, we demonstrate marked over-expression of mRNAs for the majorcomponents of NADPH oxidase (NOX1 and gp91 phox) in cisplatin-inducednephrotoxicty. Over-expression of these oxidative marker genes furtheraccompanied by increased ROS generation evident from the elevated kidneylipid per-oxidation in the cisplatin-administered rat. We alsodemonstrate reduced renal SOD1 and SOD3 expressions, and suggest thatsuch reduction contributes to the oxidative stress in cisplatinadministered rat. Similar observations are reported in earlier studieswhere cisplatin-induced nephropathy is accompanied by increased MDAlevel and elevated expression and activity of NADPH oxidase.Interestingly, our study also demonstrate that EET analogs markedlyreduced the renal oxidative stress by reducing the renal lipidper-oxidation, marked reductions in the expression of the major NADPHoxidase subunits, and also by increased expression of SOD1. Indeed, in arecent study it is reported that EET up-regulates the expression andactivity of SOD during toxic insult, thus enhance ROS scavenging andreduce oxidative stress. Similar to our findings, in anotherpathological model characterized with renal injury, EET mediatedreduction in oxidative stress and renal injury has been reported. Apartfrom oxidative stress, we also demonstrate that cisplatin-inducednephrotoxicity is further accompanied by elevated renal inflammatoryresponse and supports earlier evidences on important role forinflammatory mechanisms in the pathogenesis of cisplatin-inducednephrotoxicity. Indeed, cisplatin induces increased renal expression ofa variety of inflammatory chemokines and cytokines, such as TNF-α andIL-1β.

We further demonstrate that EET analog treatment reduced renalexpression of these inflammatory markers in cisplatin-inducednephrotoxicity. Our data support earlier reports of anti-inflammatoryactivity of EET that has been implicated in EET mediated organprotection in a number of pathologies characterized with organ injury.For instance, increased bioavailability of EET by sEH inhibitionprovides kidney protection in streptozotocin-induced diabetes. Moreover,over-expression of the EET producing enzyme CYP2J2 markedly protectedkidney in a chronic renal failure model of 5/6 nephrectomy. Thus, ourdata clearly indicate that along with marked reduction in oxidativestress, attenuation of cisplatin-induced renal inflammatory responses isanother mechanism by which EET analog protected kidney fromcisplatin-induced nephrotoxicity.

We have further investigated EET analog's effect on cisplatin-inducedendoplasmic reticulum (ER) stress. There is evidence that ER is one ofthe sub-cellular targets of toxins and play important role inxenobiotic-induced nephrotoxicity. In the present study we examined therenal expression of caspase 12 and GRP78 (glucose-regulated protein 78)mRNAs to investigate the involvement of the ER stress incisplatin-induced nephrotoxicity. GRP78 is considered one of thehallmarks of ER stress, while caspase 12 is an ER-specific caspase thatis activated by ER stress and specifically participates in ERstress-induced apoptosis. We observe marked up-regulation in the renalexpression of these ER stress markers that is attenuated by EET analogtreatment. Our study supports earlier observations thatcisplatin-induced nephrotoxicity is associated with ER stress. Mostimportantly, the present study also provided an interesting and novelfinding regarding the biological actions of EET, and demonstrates animportant aspect on the therapeutic potential of this lipid mediator intreating cisplatin-induced nephrotoxicity.

Cisplatin and other drug-related nephrotoxicity is associated withapoptosis¹⁰ that is caused by elevated oxidative stress, inflammationand ER stress. It is reported that during cisplatin-inducednephrotoxicity, the cellular stress caused by oxidative stress,inflammation and ER stress leads to a reduction of anti-apoptotic Bcl2and activation of the pro-apoptotic Bcl2 family proteins like the Bcl-2associated X protein (Bax) and Bcl-2 antagonist/killer protein (Bak) inthe kidney. This enhanced pro-apoptotic signaling leads to theactivation of caspase 3 followed by apoptosis of the renal cells. Here,we demonstrate that EET analog treatment protect the kidney fromcisplatin-induced cell death by increasing the expression ofanti-apoptotic Bcl2 and reducing the pro-apoptotic Bak/Bcl2 and Bax/Bcl2ratios along with a marked reduction in caspase 3 activity.

We also demonstrate an EET analog mediated attenuation in the renalexpression of caspase 12 that plays an essential role in ER stressmediated apoptosis. Indeed, it is earlier reported that EET attenuatesseveral major apoptotic events including elevated Bcl2 protein mediatedpro-apoptotic signaling and caspase 3 activity. These observationssupport our view on EET analog's ability to reduce renal cell death incisplatin-induced nephrotoxicity through its effect on the Bcl2proteins, ER stress specific caspase 12 and on the apoptosis executionercaspase, caspase 3.

We have clearly demonstrated that EET analog treatment providesprotection from cisplatin-induced nephrotoxicity through multiplemechanisms, and strongly indicate a possible therapeutic promise.However, it is important that before the clinical use of newcytoprotective agents, not only protection from toxicity, but also theabsence of an interference of the agent with the anti-cancer activity ofthe cytotoxic agents used is demonstrated. To this end, in an in vitroapproach we have investigated whether in vitro exposure of normal kidneycells (HEK293) or several human cancer cell-lines (Hela, NCCIT, U87) tovarious concentrations of an EET analog (EET-A) influence cell growth orthe cytotoxic effect of cisplatin. Considering the comparable kidneyprotective effects of the two EET analogs used in this study, we havechosen one EET analog for this particular experiment. We demonstratedthat in the presence and absence of EET analog (EET-A) cisplatin isequally potent in exerting its chemotherapeutic effect. Moreover, wehave also investigated if EET-A influence the growth of any of thecancer lines used in this study, and clearly demonstrated that EET-A hadno effect on the growth of any of these cancer cell lines.

In conclusion, we have provided strong evidence that the kidneyprotective effect of the above-identified EET analogs in drug-inducednephrotoxicitn, including cisplatin-induced nephrotoxicity. We havedemonstrated that these EET analogs offered kidney protection by theinhibition of multiple signaling pathways that critically involve in thepatho-physiology of cisplatin-induced nephrotoxicity. This studyhighlighted several important biological actions of novel EET analogs interms of their anti-oxidative, anti-inflammatory, anti-ER stress andanti-apoptotic activities. The results of the current study strengthenour view on the therapeutic promise of these novel EET analogs intreating cisplatin-induced nephrotoxicity without compromisingcisplatin's chemotherapeutic potential.

While this invention has been described in conjunction with the variousexemplary embodiments outlined above, various alternatives,modifications, variations, improvements and/or substantial equivalents,whether known or that are or may be presently unforeseen, may becomeapparent to those having at least ordinary skill in the art.Accordingly, the exemplary embodiments according to this invention, asset forth above, are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinvention. Therefore, the invention is intended to embrace all known orlater-developed alternatives, modifications, variations, improvements,and/or substantial equivalents of these exemplary embodiments. Alltechnical publications, patents and published patent applications citedherein are hereby incorporated by reference in their entirety for allpurposes.

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What is claimed is:
 1. A compound selected from the group consisting of


2. The compound of claim 1, wherein the structure is


3. The compound of claim 1, wherein the structure is


4. A compound selected from the group consisting of


5. A composition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 6. A method of reducing hypertension in a subject,comprising administering to a subject a therapeutically effective amountof a compound according to claim 1, wherein hypertension in said subjectis reduced.
 7. A method of reducing nephrotoxicity in a subject,comprising administering to a subject a therapeutically effective amountof a compound according to claim 1, wherein nephrotoxicity in saidsubject is reduced.
 8. The method of claim 7, wherein the nephrotoxicityis drug-induced.
 9. The method of claim 8, wherein the nephrotoxicity iscisplatin-induced.
 10. A method of reducing cisplatin nephrotoxicity ina subject, comprising administering to a subject a therapeuticallyeffective amount of a compound according to claim 1, wherein cisplatinnephrotoxicity in said subject is reduced.
 11. A composition comprisinga compound of claim 4 and a pharmaceutically acceptable carrier.
 12. Amethod of reducing hypertension in a subject, comprising administeringto a subject a therapeutically effective amount of a compound accordingto claim 4, wherein hypertension in said subject is reduced.
 13. Amethod of reducing nephrotoxicity in a subject, comprising administeringto a subject a therapeutically effective amount of a compound accordingto claim 4, wherein nephrotoxicity in said subject is reduced.
 14. Themethod of claim 13, wherein the nephrotoxicity is drug-induced.
 15. Themethod of claim 14, wherein the nephrotoxicity is cisplatin-induced. 16.A method of reducing cisplatin nephrotoxicity in a subject, comprisingadministering to a subject a therapeutically effective amount ofcompound according to claim 4, wherein cisplatin nephrotoxicity in saidsubject is reduced.